Poly(ADP-ribose) polymerase 1 is overexpressed in nasopharyngeal carcinoma and its
inhibition enhances radiotherapy
Jeremy P.H. Chow∆,1, Wing Yu Man∆,1, Mao Mao1, Han Chen1, Florence Cheung2, John
Nicholls3, Sai Wah Tsao4, Maria Li Lung5, and Randy Y.C. Poon*
(∆) These authors contributed equally
(1) Division of Life Science, Center for Cancer Research, and State Key Laboratory of
Molecular Neuroscience, The Hong Kong University of Science and Technology, Clear
Water Bay, Hong Kong
(2) Department of Pathology, Pamela Youde Nethersole Eastern Hospital, Hong Kong
(3) Department of Pathology, (4) Department of Anatomy, (5) Department of Clinical
Oncology and Center for Cancer Research, The University of Hong Kong, Hong Kong
(*) Corresponding author: Division of Life Science, Hong Kong University of Science and
Technology, Clear Water Bay, Hong Kong
Tel: [852]-23588703
Fax: [852]-23581552
Email: [email protected]
Home page: http://ihome.ust.hk/~rycpoon
Key words: AZD2281; DNA damage; DNA repair; Olaparib; PARP1
Running title: Poly(ADP-ribose) polymerase in NPC
Grant information: This work was supported in part by the Research Grants Council grant
AOE-MG/M-08/06 to R.Y.C. Poon.
Conflict of interest: The authors declare no conflict of interest
on July 10, 2018. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 26, 2013; DOI: 10.1158/1535-7163.MCT-13-0010
2
ABSTRACT
Nasopharyngeal carcinoma (NPC) is a rare but highly invasive cancer. As options of agents
for effective combination chemoradiotherapy of advanced NPC are limited, novel therapeutic
approaches are desperately needed. The ubiquitin ligase CHFR is known to target
poly(ADP-ribose) polymerase 1 (PARP1) for degradation and is epigenetically inactivated in
NPC. We present evidence that PARP1 protein is indeed overexpressed in NPC cells in
comparison to immortalized normal nasopharyngeal epithelial cells. Tissue microarray
analysis also indicated that PARP1 protein is significantly elevated in primary NPC tissues,
with strong correlation with all stages of NPC development. We found that the PARP
inhibitor AZD2281 (Olaparib) increased DNA damage, cell cycle arrest, and apoptosis in
NPC cells challenged with ionizing radiation or temozolomide. Isobologram analysis
confirmed that the cytotoxicity triggered by AZD2281 and DNA damaging agents was
synergistic. Finally, AZD2281 also enhanced the tumor-inhibitory effects of ionizing
radiation in animal xenograft models. These observations implicate that PARP1
overexpression is an early event in NPC development and provide a molecular basis of using
PARP inhibitors to potentiate treatment of NPC with radiotherapy and chemotherapy.
on July 10, 2018. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 26, 2013; DOI: 10.1158/1535-7163.MCT-13-0010
3
INTRODUCTION
Nasopharyngeal carcinoma (NPC) is a highly invasive cancer. Although NPC is relatively
rare in most parts of the world (annual incidence rate generally less than one per 100,000
population), high incidence rates are found in southern China and Southeast Asia (1). For
example, NPC among women and men in Hong Kong is 7 and 18 per 100,000, respectively
(2). The prevailing view is that infection by Epstein-Barr virus (EBV) is a significant
etiological factor for NPC (reviewed in (3)). Epidemiological evidence also links the
consumption of dietary nitrosamines (such as salted fish) to NPC (reviewed in (4)).
Radiotherapy has been used as the primary treatment of NPC (5). This reflects
both the inaccessible location of the nasopharynx as well as the radiosensitivity of NPC
cells. More recent advances in radiotherapy such as 3D conformal radiotherapy and
intension-modulated radiotherapy have helped to achieve good prognosis, in particular
for early stage NPC (6, 7). For advanced NPC, however, treatment outcomes have been
poor due to distant metastases and recurrence (8). Chemotherapy is used in combination
with radiotherapy to control advanced NPC. Commonly used chemotherapeutic agents
are restricted to standard compounds such as the DNA-damaging agent cisplatin and the
thymidylate synthase inhibitor 5-flurouracil. Effective agents for combination
chemoradiotherapy for NPC are still to be established.
One of the earliest responses to DNA damage is the binding of poly(ADP-ribose)
polymerase (PARP) to damaged sites. PARP catalyzes the transfer and polymerization of
ADP ribose units from NAD+ to form branched polymers of ADP ribose covalently
linked to heterologous acceptor proteins or PARPs themselves (reviewed in (9)). PARP-
catalyzed poly(ADP-ribosyl)ation plays pivotal roles in the processing and resolution of
DNA breaks. The binding of PARP to the site of DNA damage activates its catalytic
on July 10, 2018. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 26, 2013; DOI: 10.1158/1535-7163.MCT-13-0010
4
activity and promotes local poly(ADP-ribose)-dependent recruitment of DNA repair
enzymes. Poly(ADP-ribosyl)ation of histones and other proteins may also mediate the
decondensation of chromatin structure required for efficient DNA repair. PARP1 is the
most abundant and active enzyme of the PARP family; but roles of other members
including PARP2 and PARP3 in DNA damage responses are emerging (10).
PARP inhibitors have been evaluated in clinical trials either as single agents or in
combination with DNA damaging agents (reviewed in (11)). As stand alone agents,
PARP inhibitors have been used to induce synthetic lethality in homologous repair-
deficient tumors. For example, PARP inhibitors are effective in targeting BRCA1- or
BRCA2-deficient breast and ovarian cancer. The rationale of the approach is that
inhibition of PARP blocks the repair of spontaneous single-strand lesions, leading to the
formation of double strand breaks through mechanisms such as stalled replication forks.
These double-strand breaks become lethal in cells that are deficient in double-strand
break repair (such as those with mutations in BRCA1 or BRCA2). Synthetic lethality of
PARP inhibitors is not limited to BRCA1 or BRCA2 deficiency. Recently, it was
discovered that PARP inhibitors markedly sensitize Ewing’s sarcoma cells harboring the
EWS-FLI1 gene translocation (12). More importantly, PARP inhibitors could potentially
be used as agents that enhance chemotherapy- or radiation-induced DNA damage in
patients without defined gene mutations. This remains an emerging field with many
parameters to be deciphered (reviewed in (11)).
While PARP1 has been implicated as a therapeutic target in several types of
cancer, its role has not been vigorously investigated in NPC. Here we present evidence
that PARP1 is overexpressed in NPC in comparison to normal nasopharyngeal cells.
Importantly, NPC cell growth was inhibited synergistically by DNA damaging agents
on July 10, 2018. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 26, 2013; DOI: 10.1158/1535-7163.MCT-13-0010
5
and an inhibitor of PARP.
on July 10, 2018. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 26, 2013; DOI: 10.1158/1535-7163.MCT-13-0010
6
MATERIALS AND METHODS
Materials
Olaparib (AZD2281) (Selleck Chemicals, Houston, TX, USA) and temozolomide (TMZ)
(Sigma-Aldrich, St. Louis, MO, USA) were obtained from the indicated suppliers.
Cell culture
IMR90 (normal fibroblasts) were obtained from the American Type Culture Collection
(Manassas, VA, USA). NPC cell lines C666-1 (13), CNE2 (14), HNE1 (15), and HONE1
(15) were obtained from NPC AoE Cell Line Repository and were propagated in RPMI1640
(for C666-1) or DMEM (for other NPC cells) supplemented with 10% (v/v) fetal bovine
serum (Life Technologies, Carlsbad, CA, USA) and 50 U/ml penicillin-streptomycin (Life
Technologies). No authentication was done by the authors. Telomerase-immortalized
nasopharyngeal epithelial cell lines NP361, NP460, and NP550 (16) were propagated in
keratinocyte serum-free medium supplemented (Life Technologies, Carlsbad, CA, USA)
with 50% v/v Epilife (Sigma, St Louis, MO, USA). Cells were grown in humidified
incubators at 37°C in 5% CO2.
Trypan blue analysis was performed as described (17). For clonogenic survival
assays, 500 cells were seeded onto 60 mm-dishes. After 16 h, the cells were irradiated with
different doses of IR and either mock-treated or exposed to the different concentrations of
AZD2281 for another 24 h. After 10 days, colonies were fixed with methanol/acetic acid
(2:1 v:v) and visualized by staining with 2% (w/v) crystal violet in 20% methanol.
on July 10, 2018. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 26, 2013; DOI: 10.1158/1535-7163.MCT-13-0010
7
Flow cytometry
Flow cytometry analysis after propidium iodide staining was performed as described
previously (17).
Ionizing radiation
IR was delivered with a caesium137 source from a MDS Nordion Gammacell 1000 Elite
Irradiator. Unless stated otherwise, cells were irradiated with a dose of 15 Gy.
γ-H2AX staining
Cells grown on poly-L-lysine-treated coverslips were fixed by ice-cold methanol for 10 min.
The cells were then washed twice with PBS, 5 min each, before permeabilized and blocked
with 3% bovine serum albumin (BSA) and 0.2% Tween-20 in PBS at room temperature for
30 min. The cells were washed twice with wash buffer (0.2% Tween-20 in PBS) before
incubated with phosphor-histone H2AXSer139 antibody (Bethyl Laboratory, Montgomery, TX,
USA) (diluted in 3% BSA, 0.2% Tween-20 with PBS) at 4°C for 16 h. The cells were then
washed with wash buffer for four times, 5 min each, and incubated in dark with Alexa Fluor
594 goat anti-rabbit IgG secondary antibodies (Life Technologies) at 25°C for 2 h. After
washed four times with wash buffer for 5 min each, the cells were stained with Hoechst
33342 (5 µg/ml in wash buffer) for 5 min, washed three times with wash buffer, before
mounted with 100 mM N-propyl-gallate in 9:1 v/v glycerol:PBS.
Isobologram analysis
The combination effects of two treatments were analyzed according to the median-effect
method of Chou and Talalay (18). Each dose-response curve was used to calculate the
on July 10, 2018. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 26, 2013; DOI: 10.1158/1535-7163.MCT-13-0010
8
isobologram, the value of median-effect dose (Dm), the sigmoidicity of the dose-effect curve
(m), and the linear correlation coefficient of the median-effect plot (r). The combination
index (CI) at different effective dose (ED) ED50, ED75 and ED90 was calculated according to
Chou and Talalay (18) using Calcusyn Version 2.1 (Biosoft, Cambridge, UK). CI value < 1
indicates synergistic effect (0.1–0.5 strong synergism; < 0.1 very strong synergism); CI value
of 1 indicates additive effect; and CI value > 1 indicates antagonistic effect.
RT-PCR
Total RNA was extracted from ~3x106 cells using Nucleospin RNA (Macherey-Nagel,
Düren, Germany). The total RNA (1 µg) was used to synthesize cDNA using High-Capacity
cDNA Reverse Transcription Kit (Life Technologies) according to the manufacturer’s
instruction. Semi-quantitative real-time PCR was performed by mixing the cDNA with
SYBR Green Real-Time PCR Master Mixes in a 7500 Fast Real-Time PCR System (Life
Technologies) using the following primers for actin (5'-GGGAAATCGTGCGTGACATT-3'; 5'-
GGAACCGCTCATTGCCAAT-3'), CHFR (5'-GGCAACCAGAGGTTTGACAT-3'; 5'-
AGTCAGGACGGGATGTTACG-3'), and PARP1 (5'-GATGGGTTCTCTGAGCTTCG-3'; 5'-
ACACCCCTTGCACGTACTTC-3').
Antibodies and immunoblotting
Antibodies against β-actin (Sigma-Aldrich, St. Louis, MO, USA), phosphor-histone
H2AXSer139 (Bethyl Laboratory, Montgomery, TX, USA), cleaved PARP1(Asp214) (BD
Biosciences, Franklin Lakes, NJ, USA), and PARP1 (sc-7150, Santa Cruz Biotechnology,
Santa Cruz, CA, USA) were obtained from the indicated suppliers. Rabbit antibodies against
on July 10, 2018. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 26, 2013; DOI: 10.1158/1535-7163.MCT-13-0010
9
CHFR were raised against a bacterially expressed GST-CHFR(C∆427) protein.
Immunoblotting was performed as described (19).
NPC tissue microarray analysis
A tissue microarray containing normal nasopharyngeal epithelium and NPC specimens
from Hong Kong was constructed. The NPC specimens were obtained from NPC
patients prior to treatment at Queen Mary Hospital and Pamela Youde Nethersole Eastern
Hospital. Construction of the NPC tissue microarray and immunohistochemical staining
using the standard streptavidin-biotin-peroxidase complex method was similar to that
previously reported (20). Slides were incubated with antibodies against PARP1 at 1:200
dilution and counterstained with hematoxylin. The immunohistochemical stained tissue
microarray was reviewed by two independent pathologists. For evaluation of the
staining, the nonmalignant and malignant tissues were scored by assessing the PARP1
staining in the nucleus. A random four views were counted under 400x microscopy. The
intensity of staining was graded by an arbitrary scale that ranged from 0 to 3,
representing negative (“0”), weak (“1”), moderate (“2”) and strong (“3”) staining,
respectively. Staining values of 0 and 1 were classified as low expression, while 2 and 3
were classified as high expression. The Pearson χ2 test was used to assess the association
between PARP1 expression and several clinicopathologic variables. A p value <0.05 was
regarded as statistically significant.
Tumor xenografts
The experimental protocol was evaluated and approved by the Animal Care Committee,
HKUST. HONE1 cells (2x107) were injected subcutaneously into both sides of the dorsa of
on July 10, 2018. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 26, 2013; DOI: 10.1158/1535-7163.MCT-13-0010
10
4-6-week-old female BALB/c nude mice. Four animals (n=8) per group were used in control
and combination treatment and three animals were used in the single treatments. Tumors
were regularly measured using a Vernier caliper. Volume was calculated according to the
formula: π/6×length×width2. Day=0 was designated as when the tumor volume was ~100
mm3. AZD2281 (50 mg/kg) was administrated with intraperitoneal injection into the mice
daily from day 1 to day 5. IR (2 Gy) was delivered at day 1 and 4. In the combination
treatment, AZD2281 was administrated 30 min before IR delivery. Mice were killed when
the tumors in control group reached 1,000 mm3.
on July 10, 2018. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 26, 2013; DOI: 10.1158/1535-7163.MCT-13-0010
11
RESULTS
Overexpression of PARP1 protein in NPC cell lines
Frequent alterations of gene expression have been reported in NPC. However, their
implication in tumorigenesis or treatment is not always immediately apparent. Among these,
transcription of the ubiquitin ligase CHFR was found to be epigenetically inactivated in NPC
through promoter methylation (21). To study if CHFR protein is altered in NPC cells,
antibodies against CHFR were generated. Lysates from different NPC cell lines (C666-1,
CNE2, HNE1, and HONE1) were prepared and analyzed with immunoblotting using
antibodies specific for CHFR. Normal nasopharyngeal (NP) epithelial cells that were
immortalized with telomerase were used as a comparison. CHFR was found to be
downregulated in all the NPC cell lines (Fig 1A). In agreement with this, CHFR was also
present at a relatively high level in three different immortalized NP epithelial cell lines
(NP361, NP460, and NP550) (Fig 1B). As a control, the normal human fibroblasts strain
(IMR90) also contained a high abundance of CHFR.
Recently, CHFR was reported to be able to regulate PARP1 by targeting PARP-1 for
ubiquitination and degradation (22). We also found that PARP1 expression could be reduced
by overexpression of CHFR and increased by knockdown of CHFR (our unpublished data).
This prompted us to examine the expression of PARP1 in NPC cells. Significantly, PARP1
displayed an inverse relationship with CHFR, expressing at high levels in all the NPC cell
lines and at low levels in the NP epithelial cell lines (Fig 1A and 1B). As expected, CHFR
mRNA was present at a lower level in NPC cell lines than in the NP epithelial cell lines (Fig
1C). Consistent with regulation by post-translational mechanisms, we found that PARP1
mRNA was expressed at similar levels between NPC and NP cell lines. Taken together,
on July 10, 2018. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 26, 2013; DOI: 10.1158/1535-7163.MCT-13-0010
12
these results indicate that PARP1 is present at a significantly higher level in NPC cell lines
than in normal NP epithelial cells.
Inhibition of PARP enhances the cytotoxicity induced by DNA damaging agents in
NPC cells
Given that PARP1 is overexpressed in NPC cells, we next addressed if this translates into
alterations in the sensitivity to PARP inhibitors. Olaparib (AZD2281) (23) is an orally active
PARP inhibitor undergoing Phase I and II clinical trials (11). Incubation of NPC and normal
NP epithelial cells with AZD2281 alone induced a stress response. In the NPC cells HONE1
and HNE1, a relatively high concentration of AZD2281 (10 µM) induced apoptotic cell
death (as indicated by the appearance of sub-G1 cells) (Fig 1D). An increase in 4N DNA
contents was also apparent, in particular for HONE1, suggesting a robust activation of the G2
DNA damage checkpoint. In contrast, no sub-G1 cells were detected when normal NP
epithelial cells were challenged with the same concentrations of AZD2281. In agreement
with this, trypan blue analysis indicated that a dose-dependent reduction of cell number for
NPC cells (Fig 1E). In contrast, only a marginal decrease in the number of viable cells was
observed after normal NP epithelial cells were incubated with AZD2281. Consistent with an
increase in apoptotic cell death in NPC cells, we found that AZD2281 stimulated an
accumulation of cleaved PARP1 and p53 in a dose-dependent manner (Fig 1F). The long
term clonogenic survival of HONE1 cells was likewise reduced by AZD2281 treatment (Fig
1G).
We next examined if inhibition of PARP could potentiate the cytotoxic effects of
DNA damaging agents. A partial G2 arrest was stimulated in both NPC and NP epithelial
cells after irradiated with 10 Gy of ionizing radiation (IR) (Fig 2A). Significantly more NPC
on July 10, 2018. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 26, 2013; DOI: 10.1158/1535-7163.MCT-13-0010
13
cells were arrested in G2 when they were irradiated in the presence of 1 µM of AZD2281. In
marked contrast, normal NP epithelial cells appeared to be impervious to AZD2281. As a
control, exposure of NPC cells to AZD2281 alone did not affect normal cell cycle
progression (see also Fig 1D). Moreover, an extensive sub-G1 population was induced when
HONE1 were treated with both IR and AZD2281. Although sub-G1 population was not
apparent for HNE1 and CNE2 cells, trypan blue exclusion analysis confirmed that
substantial cell death was triggered when the cells were exposed to both IR and AZD2281
(Fig 2B).
To ensure that the stimulation of NPC cytotoxicity by AZD2281 was not limited to
IR, cells were also treated with the DNA-methylating agent temozolomide (TMZ).
Incubation with either AZD2281 or TMZ (125 µM) alone did not significantly stimulate
apoptosis. Massive apoptosis was triggered when NPC cells were incubated with both TMZ
and AZD2281 together (Fig 3A). Although normal NP epithelial cells also responded to the
combined AZD2281 and TMZ treatment by displaying more G2 arrested cells, there was no
increase in sub-G1 population. Consistent with the results from the flow cytometry, high
level of γ-H2AX was induced after incubation with AZD2281 and TMZ in HONE1, but not
in NP460 (Fig 3B).
To see if the increase in DNA damage-mediated arrest and cell death in AZD2281-
treated cells was caused by defective DNA repair, we next examined γ-H2AX staining in the
presence or absence of AZD2281 (Fig 4A). As expected, irradiation with 5 Gy of IR
induced γ-H2AX foci formation in both NPC (HONE1) and NP epithelial (NP460) cells.
The γ-H2AX foci staining returned to a background level after 24 h, suggesting that the
damaged DNA was repaired in both NPC and NP cells (Fig 4B). Significantly, efficient
on July 10, 2018. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 26, 2013; DOI: 10.1158/1535-7163.MCT-13-0010
14
DNA repair in HONE1 cells was attenuated in the presence of AZD2281. In contrast, the
DNA repair in NP460 cells was resistant to AZD2281.
Collectively, these results indicate that inhibition of PARP enhances IR- or TMZ-
mediated DNA damage and apoptosis in NPC cells but not in normal NP epithelial cells.
PARP inhibition and ionizing radiation act synergistically to promote cytotoxicity
To investigate more vigorously if the effects of PARP inhibition and DNA damage on NPC
cells represent synergism, isobologram analysis was carried out according to the median-
effect method of Chou and Talalay (18). Using trypan blue exclusion assays to monitor
cytotoxicity, we found that AZD2281 and IR acted synergistically on NPC cells (HONE1
and HNE1) (Fig 5A and Table 1). The combination index (CI) was less than 1, which
indicated synergism, over a range of effective dose (ED50, ED75 and ED90). In contrast,
synergism between AZD2281 and IR were less effective on NP epithelial cells (NP460),
with the CI values actually exceeding 1 at ED90 (indicating antagonism). We also examined
the long term survival of HONE1 cells using clonogenic survival assays (Fig 5B and Table
2). Similar analysis indicated a synergism between AZD2281 and IR at ED75 and ED90 for
long term clonogenic survival. Collectively, these data demonstrated that AZD2281 and IR
act synergistically to induce cytotoxic effects on NPC cells.
Combined AZD2281 and ionizing radiation treatment reduces tumor growth in
tumor xenograft models
We next evaluated the effects of PARP inhibition on NPC growth in animal tumor models.
HONE1 cells were injected subcutaneously into nude mice. AZD2281, IR, or a combination
of both were delivered using a fractionated dose approach (Fig 6A). Treatment with
on July 10, 2018. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 26, 2013; DOI: 10.1158/1535-7163.MCT-13-0010
15
AZD2281 or IR individually reduced the rate of tumor growth. But more significant
reduction of tumor growth was induced in the group treated with both AZD2281 and IR.
More importantly, there was in fact a decrease in tumor volume during the period of
combined treatment (Fig 6A and 6B). Tumor size increased again only after the treatment
ceased. These results indicated that AZD2281 and IR exerted a strong tumor inhibitory
effect in xenograft mouse models.
Overexpression of PARP1 in primary human NPC
To confirm that the overexpression of PARP1 observed in NPC cell lines are relevant in
primary NPC tumors, we next examined the expression of PARP1 in primary human NPC
using tissue microarray analysis (Fig 6C). Normal NP and NPC tissues were scored for low
or high expression of PARP1 by assessing PARP1 staining in the nucleus (Table 3). High
expression of PARP1 was found in NPC tissues. Correlation was observed between PARP1
expression and several clinicopathologic parameters, including patient age and survival.
Importantly, an increase in PARP1 expression could be observed in all NPC stages,
suggesting that the alteration of PARP1 expression is an early event in NPC development.
on July 10, 2018. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 26, 2013; DOI: 10.1158/1535-7163.MCT-13-0010
16
DISCUSSION
In this study, we found that PARP1 protein is highly expressed in NPC cell lines and primary
NPC tissues in comparison to nasopharyngeal epithelial cells. The PARP inhibitor
AZD2281 increased DNA damage, cell cycle arrest, and apoptosis in NPC cell lines treated
with IR or temozolomide. Finally, AZD2281 also enhanced the tumor-inhibitory effects of
IR in mouse xenograft models.
A major focus of the clinical development of PARP inhibitors is to determine if they
can potentiate radio/chemotherapy-mediated DNA damage, especially for tumors that lack
intrinsic defects in DNA damage repair (reviewed in (11)). For NPC, we found that PARP
inhibition was indeed effective in potentiating the cytotoxicity of DNA damaging agents
using both cell line (Fig 2 and 3) and animal models (Fig 6). Isobologram analysis indicated
that at least for the cell line models, the effects of AZD2281 and IR were synergistic rather
than simply additive (Fig 5). AZD2281 and IR displayed synergism on NPC cells over the
range of effective doses in both short-term growth assay (Fig 5A and Table 1) and long-term
cell survival (Fig 5B and Table 2). Results from the xenograft studies further indicated that
combinational treatment of AZD2281 and IR could further lead to shrinkage of tumor size
(Fig 6). However, the treatment also has synergistic effects on normal NP cells (for ED50
and ED75) (Fig 5A and Table 1). Moreover, synergism was found at all effective doses for
long-term NPC cell growth (Fig 5B and Table 2). Furthermore, the combined AZD2281 and
IR treatment resulted in a ~15% weight loss of the animals (Supplemental Fig 1), suggesting
potential high toxicity to normal cells.
Hence although our findings provide a rationale to support testing of PARP inhibitors and
DNA damaging agents on NPC, further research is required to uncover the precise clinical
responses of these treatments.
on July 10, 2018. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 26, 2013; DOI: 10.1158/1535-7163.MCT-13-0010
17
Conceptually, PARP inhibition should only enhance radio/chemotherapy if DNA
damage is selectively increased in tumor compared to normal tissues. Tumors that already
harbor DNA repair defects such as mutations in BRCA1 or BRCA2 genes serve as classic
examples. So far there is no evidence of impairment of the components of the DNA repair
pathways such as BRCA2 in NPC (24, 25). As implicated by our study, however, the
abundance of PARP1 in tumors may also play a role in selectively sensitizing the cells to
PARP inhibitors. NPC cell lines contained significantly more PARP1 than NP epithelial
cells (Fig 1A and 1B) were more sensitive to AZD2281 alone (Fig 1D and 1E) or when
AZD2281 was added together with DNA damaging agents (Fig 2 and 3).
The observations that PARP1 was overexpressed even at early stages of NPC
development (Table 3) imply that alteration of PARP1 could be a driver of tumorigenesis.
Consistent with our observations, overexpression of PARP1 has also been reported in other
tumors, including colorectal cancer (26), endometrial cancer (27), hepatocellular carcinoma
(28), and melanomas (27). Several studies have reported the association of PARP1 protein
expression with a worse prognosis. For example, PARP1 overexpression is associated with
overall poor prognosis, higher tumor grade, BRCA1-mutated and triple-negative phenotype
breast cancers (29-31). In NPC, PARP1 expression was correlated with increase patient age
and poorer survival (Table 3).
What are the mechanisms that lead to PARP1 overexpression in NPC and other
cancers? Transcription of the PARP1 gene relies on transcription factors such as Sp1 and
Sp3 (32) and NF1 (33). However, the increase in PARP1 protein in NPC was unlikely to be
due to an increase in transcription because no significant variation in PARP1 mRNA
between NPC and NP epithelial cell lines was observed (our unpublished data). This is also
consistent with the lack of report on an alteration of PARP1 mRNA expression from various
on July 10, 2018. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 26, 2013; DOI: 10.1158/1535-7163.MCT-13-0010
18
microarray studies. Instead, we believe that the protein level of PARP1 is usually kept at a
low level by CHFR in normal NP cells (22). The epigenetic inactivation of CHFR in NPC
(21) provides a molecular basis of how PARP1 is upregulated at the protein level (see Fig 6D
for a model). In this connection, it is possible that methylation of the CHFR promoter can be
used as a marker for PAPR1 expression, which in turn can be used as a prediction for
sensitivity to PARP inhibitors.
An interesting question is why overexpression of PARP1 would lead to an increase in
sensitivity to PARP inhibition. We believe that “PARP1 addiction” may play a role on the
reliance of NPC cells on PARP1 for DNA damage responses. As the DNA repair circuitry
gets re-wired by the constitutive overexpression of PARP1, the cell may become exquisitely
dependent on PARP1 activity. NPC cells were clearly more dependent on PARP activity for
effective repair than NP epithelial cells (Fig 4). The PARP1 addiction may be analogous to
the addiction to many oncogenes in cancer cells. Another possible reason why PARP1-
overexpressing cells were sensitive to PARP inhibitor is the recent finding that the
cytotoxicity of PARP inhibitors including AZD2281 is in part caused the trapping of PARP1
at damaged DNA complexes (34). Hence higher expression of PARP1 may allow larger
number of PARP1-DNA complexes to form.
Results from this study introduced further questions about PARP1 and NPC.
PARP inhibitors in development mimic the nicotinamide moiety of NAD+ and bind
PARP’s catalytic domain. In addition to AZD2281, several PARP inhibitors are in the
development pipeline. It would be interesting to see if they offer better efficacy when
combined with IR in treatment of NPC. Although PARP1 is believed to be the most
abundant isoform of the PARP family, it is possible that other family members may also
play a role in NPC. Inhibitors that target specific isoforms may be useful. Eventually,
on July 10, 2018. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 26, 2013; DOI: 10.1158/1535-7163.MCT-13-0010
19
randomized studies will be needed to validate PARP inhibitors as therapeutic agents for
NPC patients. Another unresolved issue is although we know that PARP1 is
overexpressed in NPC and other cancers, little is known about its activity. It would be
interesting to assay PARP1 activity in cell lines and tumor specimens from patients
treated with PARP inhibitors. In addition to DNA repair, PARP family also participates
in other processes, including remodeling of chromatin and the regulation of transcription
(reviewed in (35)). It is conceivable that the sensitization of NPC cells with PARP
inhibition was not just due to disruption of DNA repair.
In conclusion, PARP1 overexpression is an early event in NPC development.
Cell line and animal studies revealed that PARP inhibitors might be able to potentiate
radiotherapy and chemotherapy for NPC.
on July 10, 2018. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 26, 2013; DOI: 10.1158/1535-7163.MCT-13-0010
20
ACKNOWLEDGEMENTS
We thank Anita Lau and Kenji Nishiura for technical assistance, and Junyi Zhang for help in
generating the CHFR antibodies.
on July 10, 2018. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 26, 2013; DOI: 10.1158/1535-7163.MCT-13-0010
21
REFERENCES
1. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics.
CA Cancer J Clin 2011;61:69-90.
2. Curado MP, Edwards B, Shin HR, Storm H, Ferlay J, Heanue M, et al. Cancer
incidence in five continents. Lyon: International Agency for Research on Cancer;
2007:896.
3. Dawson CW, Port RJ, Young LS. The role of the EBV-encoded latent membrane
proteins LMP1 and LMP2 in the pathogenesis of nasopharyngeal carcinoma (NPC).
Semin Cancer Biol 2012;22:144-53.
4. Chang ET, Adami HO. The enigmatic epidemiology of nasopharyngeal carcinoma.
Cancer Epidemiol Biomarkers Prev 2006;15:1765-77.
5. Chan AT, Felip E. Nasopharyngeal cancer: ESMO clinical recommendations for
diagnosis, treatment and follow-up. Ann Oncol 2009;20 Suppl 4:123-5.
6. Waldron J, Tin MM, Keller A, Lum C, Japp B, Sellmann S, et al. Limitation of
conventional two dimensional radiation therapy planning in nasopharyngeal carcinoma.
Radiother Oncol 2003;68:153-61.
7. Cheng JC, Chao KS, Low D. Comparison of intensity modulated radiation therapy
(IMRT) treatment techniques for nasopharyngeal carcinoma. Int J Cancer 2001;96:126-
31.
8. Lee AW, Poon YF, Foo W, Law SC, Cheung FK, Chan DK, et al. Retrospective
analysis of 5037 patients with nasopharyngeal carcinoma treated during 1976-1985:
overall survival and patterns of failure. Int J Radiat Oncol Biol Phys 1992;23:261-70.
9. Luo X, Kraus WL. On PAR with PARP: cellular stress signaling through poly(ADP-
ribose) and PARP-1. Genes Dev 2012;26:417-32.
on July 10, 2018. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 26, 2013; DOI: 10.1158/1535-7163.MCT-13-0010
22
10. De Vos M, Schreiber V, Dantzer F. The diverse roles and clinical relevance of PARPs
in DNA damage repair: current state of the art. Biochem Pharmacol 2012;84:137-46.
11. Kummar S, Chen A, Parchment RE, Kinders RJ, Ji J, Tomaszewski JE, et al. Advances
in using PARP inhibitors to treat cancer. BMC Med 2012;10:25.
12. Garnett MJ, Edelman EJ, Heidorn SJ, Greenman CD, Dastur A, Lau KW, et al.
Systematic identification of genomic markers of drug sensitivity in cancer cells. Nature
2012;483:570-5.
13. Cheung ST, Huang DP, Hui AB, Lo KW, Ko CW, Tsang YS, et al. Nasopharyngeal
carcinoma cell line (C666-1) consistently harbouring Epstein-Barr virus. Int J Cancer
1999;83:121-6.
14. Sizhong Z, Xiukung G, Yi Z. Cytogenetic studies on an epithelial cell line derived from
poorly differentiated nasopharyngeal carcinoma. Int J Cancer 1983;31:587-90.
15. Glaser R, Zhang HY, Yao KT, Zhu HC, Wang FX, Li GY, et al. Two epithelial tumor
cell lines (HNE-1 and HONE-1) latently infected with Epstein-Barr virus that were
derived from nasopharyngeal carcinomas. Proc Natl Acad Sci U S A 1989;86:9524-8.
16. Li HM, Man C, Jin Y, Deng W, Yip YL, Feng HC, et al. Molecular and cytogenetic
changes involved in the immortalization of nasopharyngeal epithelial cells by
telomerase. Int J Cancer 2006;119:1567-76.
17. Siu WY, Arooz T, Poon RY. Differential responses of proliferating versus quiescent
cells to adriamycin. Exp Cell Res 1999;250:131-41.
18. Chou TC, Talalay P. Quantitative analysis of dose-effect relationships: the combined
effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul 1984;22:27-55.
on July 10, 2018. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 26, 2013; DOI: 10.1158/1535-7163.MCT-13-0010
23
19. Poon RY, Toyoshima H, Hunter T. Redistribution of the CDK inhibitor p27 between
different cyclin.CDK complexes in the mouse fibroblast cell cycle and in cells arrested
with lovastatin or ultraviolet irradiation. Mol Biol Cell 1995;6:1197-213.
20. Xie D, Sham JS, Zeng WF, Lin HL, Che LH, Wu HX, et al. Heterogeneous expression
and association of beta-catenin, p16 and c-myc in multistage colorectal tumorigenesis
and progression detected by tissue microarray. Int J Cancer 2003;107:896-902.
21. Cheung HW, Ching YP, Nicholls JM, Ling MT, Wong YC, Hui N, et al. Epigenetic
inactivation of CHFR in nasopharyngeal carcinoma through promoter methylation. Mol
Carcinog 2005;43:237-45.
22. Kashima L, Idogawa M, Mita H, Shitashige M, Yamada T, Ogi K, et al. CHFR protein
regulates mitotic checkpoint by targeting PARP-1 protein for ubiquitination and
degradation. J Biol Chem 2012;287:12975-84.
23. Menear KA, Adcock C, Boulter R, Cockcroft XL, Copsey L, Cranston A, et al. 4-[3-(4-
cyclopropanecarbonylpiperazine-1-carbonyl)-4-fluorobenzyl]-2H-phthalazin- 1-one: a
novel bioavailable inhibitor of poly(ADP-ribose) polymerase-1. J Med Chem
2008;51:6581-91.
24. Qin HD, Shugart YY, Bei JX, Pan QH, Chen L, Feng QS, et al. Comprehensive
pathway-based association study of DNA repair gene variants and the risk of
nasopharyngeal carcinoma. Cancer Res 2011;71:3000-8.
25. Cheng Y, Lung HL, Wong PS, Hao DC, Man CS, Stanbridge EJ, et al. Chromosome
13q12 region critical for the viability and growth of nasopharyngeal carcinoma hybrids.
Int J Cancer 2004;109:357-62.
on July 10, 2018. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 26, 2013; DOI: 10.1158/1535-7163.MCT-13-0010
24
26. Idogawa M, Yamada T, Honda K, Sato S, Imai K, Hirohashi S. Poly(ADP-ribose)
polymerase-1 is a component of the oncogenic T-cell factor-4/beta-catenin complex.
Gastroenterology 2005;128:1919-36.
27. Ghabreau L, Roux JP, Frappart PO, Mathevet P, Patricot LM, Mokni M, et al.
Poly(ADP-ribose) polymerase-1, a novel partner of progesterone receptors in
endometrial cancer and its precursors. Int J Cancer 2004;109:317-21.
28. Shimizu S, Nomura F, Tomonaga T, Sunaga M, Noda M, Ebara M, et al. Expression of
poly(ADP-ribose) polymerase in human hepatocellular carcinoma and analysis of
biopsy specimens obtained under sonographic guidance. Oncol Rep 2004;12:821-5.
29. Rojo F, Garcia-Parra J, Zazo S, Tusquets I, Ferrer-Lozano J, Menendez S, et al.
Nuclear PARP-1 protein overexpression is associated with poor overall survival in
early breast cancer. Ann Oncol 2012;23:1156-64.
30. Domagala P, Huzarski T, Lubinski J, Gugala K, Domagala W. PARP-1 expression in
breast cancer including BRCA1-associated, triple negative and basal-like tumors:
possible implications for PARP-1 inhibitor therapy. Breast Cancer Res Treat
2011;127:861-9.
31. Ossovskaya V, Koo IC, Kaldjian EP, Alvares C, Sherman BM. Upregulation of Poly
(ADP-Ribose) Polymerase-1 (PARP1) in Triple-Negative Breast Cancer and Other
Primary Human Tumor Types. Genes Cancer 2010;1:812-21.
32. Zaniolo K, Rufiange A, Leclerc S, Desnoyers S, Guerin SL. Regulation of the
poly(ADP-ribose) polymerase-1 gene expression by the transcription factors Sp1 and
Sp3 is under the influence of cell density in primary cultured cells. Biochem J
2005;389:423-33.
on July 10, 2018. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 26, 2013; DOI: 10.1158/1535-7163.MCT-13-0010
25
33. Laniel MA, Bergeron MJ, Poirier GG, Guerin SL. A nuclear factor other than Sp1
binds the GC-rich promoter of the gene encoding rat poly(ADP-ribose) polymerase in
vitro. Biochem Cell Biol 1997;75:427-34.
34. Murai J, Huang SY, Das BB, Renaud A, Zhang Y, Doroshow JH, et al. Trapping of
PARP1 and PARP2 by Clinical PARP Inhibitors. Cancer Res 2012;72:5588-99.
35. Krishnakumar R, Kraus WL. The PARP side of the nucleus: molecular actions,
physiological outcomes, and clinical targets. Mol Cell 2010;39:8-24.
on July 10, 2018. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 26, 2013; DOI: 10.1158/1535-7163.MCT-13-0010
26
TABLES Combination index (CI) Dm m r ED50 ED75 ED90 HONE1 0.510 0.633 0.786 1.037 0.940 0.998 HNE1 0.199 0.181 0.165 0.707 0.764 0.961 NP460 0.468 0.740 1.372 0.860 0.538 0.943
Table 1. AZD2281 and IR act synergistically to induce cytotoxicity in NPC cells. NPC (HONE1 and HNE1) and NP epithelial cells (NP460) were treated with IR and AZD2281 for 48 h and analyzed with trypan blue analysis. The combination index (CI) at ED50, ED75 and ED90 was calculated. The value of median-effect dose (Dm), the sigmoidicity of the dose-effect curve (m), and the linear correlation coefficient of the median-effect plot (r) are indicated. A CI value < 1 indicates synergism.
on July 10, 2018. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 26, 2013; DOI: 10.1158/1535-7163.MCT-13-0010
27
Combination index (CI) Dm m r ED50 ED75 ED90 HONE1 1.142 0.830 0.606 1.072 3.940 1.000
Table 2. AZD2281 and IR act synergistically on clonogenic survival of NPC cells. HONE1 cells were treated with IR and AZD2281 and analyzed with clonogenic survival assays. The combination index (CI) at ED50, ED75 and ED90 was calculated. A CI value < 1 indicates synergism.
on July 10, 2018. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 26, 2013; DOI: 10.1158/1535-7163.MCT-13-0010
28
PARP1 expression Low High Variable n (%) n (%) Total p
Age ≥50 (median) 29 (58) 21 (42) 50 <50 11 (24) 34 (76) 45 0.003*
Sex Male 29 (43) 38 (57) 67 Female 13 (42) 18 (58) 31 0.900 Survival Alive 19 (54) 16 (46) 35 Dead 15 (29) 36 (71) 51 0.020*
NPC stage Normal 10 (91) 1 (9) 11
I 1 (20) 4 (80) 5 0.005*
II 12 (43) 16 (57) 28 0.006* III 7 (41) 10 (59) 17 0.008* IV 13 (39) 20 (61) 33 0.003* M 3 (38) 5 (62) 8 0.013* I,II,II,IV,M 36 (40) 55 (60) 91 0.001*
Table 3. PARP1 is overexpressed in human primary NPC. The expression of PARP1 in normal NP and NPC tissues was analyzed using tissue microarray and immunohistochemistry. Nonmalignant and malignant tissues were scored for low or high expression of PARP1 by assessing PARP1 staining in the nucleus. The
Pearson χ 2 test was used to assess the association between PARP1 expression and several clinicopathologic variables. Variables with statistical significant (p<0.05) by
Pearson χ 2 test are indicated by asterisks.
on July 10, 2018. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 26, 2013; DOI: 10.1158/1535-7163.MCT-13-0010
29
FIGURE LEGENDS
Figure 1. NPC cell lines overexpress PARP1 and are sensitive to PARP1 inhibition.
(A) Overexpression of PARP1 and downregulation of CHFR in NPC cell lines. Several NPC
cell lines (C666-1, CNE2, HNE1, and HONE1) and an immortalized normal nasopharyngeal
cell line (NP460) were analyzed. Cell-free extracts were prepared and the indicated proteins
were detected by immunoblotting. Actin analysis was included to assess protein loading and
transfer.
(B) The expression of PARP1 and CHFR in several immortalized normal nasopharyngeal
cell lines (NP361, NP460, NP550) and NPC cell lines (C666-1 and HONE1) were analyzed.
Normal human fibroblasts strain IMR90 was also included as a control. Actin analysis was
included to assess protein loading and transfer.
(C) CHFR but not PARP1 is regulated at the transcriptional level in NPC cell lines. The
relative expression of the mRNA of PARP1 and CHFR in NPC cell lines (CNE2, C666-1,
HNE1) and immortalized normal nasopharyngeal cell lines (NP361, NP460, NP550) was
quantified with semi-quantitative RT-PCR and normalized with actin mRNA (mean±SD of
three independent experiments).
(D) AZD2281 promotes apoptotic cell death in NPC cells. NPC (HONE1 and HNE1) and
normal NP epithelial cells (NP460) were incubated with either control buffer or the indicated
concentrations of AZD2281 for 48 h. The cells were harvested, fixed, and their DNA
contents were analyzed with flow cytometry. The positions of 2N and 4N DNA contents are
indicated.
(E) AZD2281 reduces cell viability in NPC cells. NPC and normal NP epithelial cells were
treated as in panel C. The cells were stained with trypan blue and the number of viable cells
was analyzed with a haemocytometer.
on July 10, 2018. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 26, 2013; DOI: 10.1158/1535-7163.MCT-13-0010
30
(F) AZD2281 promotes PARP1 cleavage in HONE1. HONE1 cells were treated as in panel
C. Lysates were prepared and subjected to immunoblotting analysis. Equal loading of
lysates was confirmed by immunoblotting for actin.
(G) AZD2281 reduces clonogenic survival in HONE1 cells. HONE1 cells were incubated
with different concentrations of AZD2281 for 48 h. The cells were washed and replated for
clonogenic survival assays.
Figure 2. AZD2281 enhances IR-mediated DNA damage responses in NPC cells.
(A) AZD2281 increases IR-mediated G2 arrest and apoptosis in NPC cells. NPC (HONE1,
HNE1, and CNE2) and normal NP epithelial cells (NP460) were treated with either control
buffer, AZD2281 (1 µM), IR (10 Gy), or a combination of IR and AZD2281. The cells were
harvested after 48 h and analyzed with flow cytometry. The positions of 2N and 4N DNA
contents are indicated.
(B) AZD2281 and IR reduce cell viability in NPC cells. NPC cells (HNE1 and CNE2) were
treated with either control buffer, AZD2281 (1 µM), IR (10 Gy), or a combination of IR and
AZD2281. The cells were harvested after 48 h and the viable cell number was analyzed
using trypan blue exclusion assay.
Figure 3. AZD2281 enhances TMZ-mediated DNA damage responses in NPC cells.
(A) AZD2281 increases TMZ-mediated G2 arrest and apoptosis in NPC cells. NPC (HONE1
and C666-1) and normal NP epithelial cells (NP460 and NP361) were incubated with either
control buffer, AZD2281 (1 µM), TMZ (125 µM), or a combination of TMZ and AZD2281.
The cells were harvested after 48 h and analyzed with flow cytometry. The positions of 2N
and 4N DNA contents are indicated.
on July 10, 2018. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 26, 2013; DOI: 10.1158/1535-7163.MCT-13-0010
31
(B) AZD2281 increases TMZ-mediated DNA damage in NPC cells. NPC cells (HNE1 and
CNE2) were incubated with either control buffer, AZD2281 (1 µM), TMZ (125 µM), or a
combination of TMZ and AZD2281 for 48 h. Lysates were prepared and analyzed with
immunoblotting. Equal loading of lysates was confirmed by immunoblotting for actin.
Figure 4. Inhibition of PARP disrupts DNA damage repair in NPC cells.
(A) AZD2281 inhibits repair of IR-mediated DNA damage in NPC but not in normal NP
epithelial cells. NPC (HONE1) and NP epithelial cells (NP460) were irradiated with IR (5
Gy), either the absence or presence of AZD2281 (1 µM). The cells were fixed at either 1 h
or 24 h after irradiation (cells treated with AZD2281 alone were also harvested after 24 h).
DNA damage and recovery were assessed by immunostaining with an antibody against γ-
H2AX. Nuclei were identified by staining with Hoechst 33342 dye. Representative images
are shown the various treatments.
(B) Quantification of γ-H2AX foci in the presence or absence of AZD2281. Cells were
treated as described in panel A. The percentage of cells positive for γ-H2AX was quantified
(n=75). Cells containing more than five γ-H2AX foci in the nucleus were considered as
positive. Mean±SD of three independent experiments.
Figure 5. AZD2281 and IR act synergistically to induce cytotoxicity in NPC cells.
(A) AZD2281 and IR act synergistically on NPC cells but not NP460 in reducing cell
number. NPC (HONE1 and HNE1) and NP epithelial cells (NP460) were treated with IR
and AZD2281 for 48 h and analyzed with trypan blue analysis. Isobologram plots of
on July 10, 2018. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 26, 2013; DOI: 10.1158/1535-7163.MCT-13-0010
32
effective dose (ED) ED90 are shown. Summary of the combination index (CI) is shown in
Table 1.
(B) AZD2281 and IR act synergistically on clonogenic survival of NPC cells. HONE1 cells
were treated with IR and AZD2281 and analyzed with clonogenic survival assays.
Isobologram plots of effective dose ED50, ED75, and ED90 are shown. Summary of the
combination index (CI) is shown in Table 2.
Figure 6. AZD2281 and IR inhibit tumor growth in mouse xenograft models.
(A) HONE1 cells were injected subcutaneously into nude mice. AZD2281 (closed arrow
head) and IR (opened arrow head) were delivered at the indicated time points as described in
Materials and Methods. The volume of the tumor was measured on different days (control,
white square, n=8; AZD2281, white circle, n=6; IR, grey circle, n=6; IR and AZD2281, dark
circle, n=8) (mean±SD).
(B) Representative mice with different treatments at day 28 are shown.
(C) PARP1 is overexpressed in human primary NPC. The expression of PARP1 in normal
NP and NPC tissues was analyzed using tissue microarray and immunohistochemistry.
Representative images of PARP1 and hematoxylin and eosin staining in normal NP and NPC
tissues are shown.
(D) Model of the roles of CHFR and PARP1 in NPC. In NPC, CHFR is epigenetically
inactivated by promoter hypermethylation. The decrease in CHFR protein lead to a
stabilization of PARP1 protein. As PARP1 is increased in early stages of NPC, the sustained
increase in PARP1 may contribute to tumor progression of this cancer. Moreover, PARP1
overexpression may lead to “PARP1 addiction”, in which DNA repair becomes more reliant
on PARP1 than in normal cells.
on July 10, 2018. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 26, 2013; DOI: 10.1158/1535-7163.MCT-13-0010
on July 10, 2018. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 26, 2013; DOI: 10.1158/1535-7163.MCT-13-0010
on July 10, 2018. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 26, 2013; DOI: 10.1158/1535-7163.MCT-13-0010
on July 10, 2018. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 26, 2013; DOI: 10.1158/1535-7163.MCT-13-0010
on July 10, 2018. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 26, 2013; DOI: 10.1158/1535-7163.MCT-13-0010
on July 10, 2018. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 26, 2013; DOI: 10.1158/1535-7163.MCT-13-0010
on July 10, 2018. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 26, 2013; DOI: 10.1158/1535-7163.MCT-13-0010
Published OnlineFirst August 26, 2013.Mol Cancer Ther Jeremy P.H. Chow, Wing Yu Man, Mao Mao, et al. radiotherapynasopharyngeal carcinoma and its inhibition enhances Poly(ADP-ribose) polymerase 1 is overexpressed in
Updated version
10.1158/1535-7163.MCT-13-0010doi:
Access the most recent version of this article at:
Material
Supplementary
http://mct.aacrjournals.org/content/suppl/2013/08/27/1535-7163.MCT-13-0010.DC1
Access the most recent supplemental material at:
Manuscript
Authoredited. Author manuscripts have been peer reviewed and accepted for publication but have not yet been
E-mail alerts related to this article or journal.Sign up to receive free email-alerts
Subscriptions
Reprints and
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Permissions
Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)
.http://mct.aacrjournals.org/content/early/2013/08/24/1535-7163.MCT-13-0010To request permission to re-use all or part of this article, use this link
on July 10, 2018. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 26, 2013; DOI: 10.1158/1535-7163.MCT-13-0010