International Journal of

Molecular Sciences

Article
Synergetic Effects of PARP Inhibitor AZD2281 and
Cisplatin in Oral Squamous Cell Carcinoma in Vitro
and in Vivo
Masaaki Yasukawa 1 , Hisako Fujihara 1,2, *, Hiroaki Fujimori 3,4 , Koji Kawaguchi 1 ,
Hiroyuki Yamada 5 , Ryoko Nakayama 6 , Nanami Yamamoto 1 , Yuta Kishi 1 , Yoshiki Hamada 1
and Mitsuko Masutani 3,4
1 Department of Oral and Maxillofacial Surgery, School of Dental Medicine, Tsurumi University,
2-1-3 Tsurumi, Tsurumi-ku, Yokohama, Kanagawa 230-8501, Japan;
[email protected] (M.Y.); [email protected] (K.K.);
[email protected] (N.Y.); [email protected] (Y.K.); [email protected] (Y.H.)
2 Department of Oral Hygiene, Tsurumi Junior College, 2-1-3 Tsurumi, Tsurumi-ku, Yokohama,
Kanagawa 230-8501, Japan
3 Division of Chemotherapy and Translational Research, National Cancer Center Research Institute,
5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan; [email protected] (H.F.); [email protected] (M.M.)
4 Department of Frontier Life Science, Graduate School of Biochemical Science, Nagasaki University,
1-7-1 Sakamoto, Nagasaki 852-8588, Japan; [email protected]
5 Division of Maxillofacial Surgery, Department of Oral and Maxillofacial Surgery, School of Dentistry,
Iwate Medical University 19-1 Uchimaru, Morioka Iwate 020-8050, Japan; [email protected]
6 Department of Pathology, School of Dental Medicine, Tsurumi University, 2-1-3 Tsurumi, Tsurumi-ku,
Yokohama, Kanagawa 230-8501, Japan; [email protected]
* Correspondence: [email protected]; Tel.: +81-45-580-8330; Fax: +81-45-581-1391

Academic Editor: William Chi-shing Cho

Received: 14 December 2015; Accepted: 17 February 2016; Published: 24 February 2016

Abstract: Cisplatin is a commonly used chemotherapeutic drug for treatment of oral carcinoma, and
combinatorial effects are expected to exert greater therapeutic efficacy compared with monotherapy.
Poly(ADP-ribosyl)ation is reported to be involved in a variety of cellular processes, such as
DNA repair, cell death, telomere regulation, and genomic stability. Based on these properties,
poly(ADP-ribose) polymerase (PARP) inhibitors are used for treatment of cancers, such as BRCA1/2
mutated breast and ovarian cancers, or certain solid cancers in combination with anti-cancer drugs.
However, the effects on oral cancer have not been fully evaluated. In this study, we examined
the effects of PARP inhibitor on the survival of human oral cancer cells in vitro and xenografted
tumors derived from human oral cancer cells in vivo. In vitro effects were assessed by microculture
tetrazolium and survival assays. The PARP inhibitor AZD2281 (olaparib) showed synergetic effects
with cisplatin in a dose-dependent manner. Combinatorial treatment with cisplatin and AZD2281
significantly inhibited xenografted tumor growth compared with single treatment of cisplatin or
AZD2281. Histopathological analysis revealed that cisplatin and AZD2281 increased TUNEL-positive
cells and decreased Ki67- and CD31-positive cells. These results suggest that PARP inhibitors have
the potential to improve therapeutic strategies for oral cancer.

Keywords: PARP inhibitor; oral cancer; xenografted tumor; cisplatin

1. Introduction
The incidence of oral cancer is 1%–2% of all cancers, and an estimated 300,000 new cases
were diagnosed and 145,000 patients died from oral cavity cancer (including lip cancer) in 2012
worldwide [1]. The overall incidence of oral cancer has significantly decreased over the past several

Int. J. Mol. Sci. 2016, 17, 272; doi:10.3390/ijms17030272 www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2016, 17, 272 2 of 19

decades, reflecting decreases in the consumption of tobacco and alcohol despite increased oral cancer
incidence associated with oncogenic human papilloma virus [2,3]. In Japan, the incidence of cancer in
oral cavity and pharynx is reported to be 7.2 out of 100,000 people (approximately 2% of all cancers)
and it has been slightly increasing according to the cancer registries database [4]. Current strategies for
oral cancer therapy include three major treatments: surgery, chemotherapy, radiotherapy, and their
combinations [5]. The strategies for oral cancer therapy in Tsurumi University Hospital is generally
decided based on NCCN (National Comprehensive Cancer Network) guidelines version 2, 2013 [6].
The first choice for primary treatment is always surgery, then, chemotherapy using cisplatin and
fluorouracil or radiotherapy, or a combination of them, is chosen for adjuvant therapy. In recent years,
new diagnosis techniques and treatments of oral cancer have been developed, however overall survival
has not significantly decreased in the past three decades. The reason is considered to be alterations in
tumor suppressor genes and changes in signaling pathways, leading to therapeutic resistance [7]. To
solve these problems, studies have focused on new therapeutic strategies, including combinatorial
drug regimens for cancer treatment. Such treatments are expected to exert synergetic effects resulting
in greater therapeutic effects than single drug administration, decreased side-effects, and prevention of
drug resistance. Based on these concepts, molecular-targeted drugs for cancer are of current research
interest. For oral cancer therapy, cetuximab, which targets the epidermal growth factor receptor (EGFR)
is available for clinical use. Cetuximab is activated by the ligands of EGF (epidermal growth factor)
and transforming growth factor-α, and plays a crucial role in the growth and survival of many types
of human cancers [8]. However, the overall response rate of oral cancer to cetuximab remains 36% [9].
On the other hand, poly(ADP-ribose) polymerase (PARP) inhibitors are another type of
molecular-targeted drug [10]. They have been evaluated for (1) single treatment of breast
cancer with mutations of the BRCA1/2 gene that encodes protein involved in homologous
recombination (HR) repair [11,12]; and (2) combinatorial treatments with radiotherapy or conventional
chemotherapy [11–13]. PARP-1 is an important enzyme for base excision repair (BER) [14], and loss
of PARP activity indirectly promotes accumulation of DNA double-strand breaks [15]. Therefore,
BRCA1/2-mutated cancers treated with PARP inhibitor show synthetic lethality by the severe defect
in DNA repair. PARP inhibitors are also reported to enhance the cytotoxicity of conventional
chemotherapy, such as cisplatin in breast and ovarian cancers [13]. In vitro assessments were also
reported using lymphoma, prostate cancer, and glioblastoma cells [16]. The mechanism of cisplatin is
its binding to DNA and causing inter- and intra-strand cross-links, leading to DNA template defects
and arrest of DNA synthesis and replication, especially in cancer cells [17]. Although the combination
of cisplatin and PARP inhibitors has been evaluated in several types of cancer cells [18,19], to the best
of our knowledge, it has not been evaluated in cells derived from oral cancers in vitro or in vivo.
In this study, we show that PARP-1 inhibitor AZD2281 has synergetic effects with cisplatin in vitro
and enhances suppressive effects against the growth of xenografted tumors in vivo. Our results would
provide a favorable possibility of administration of PARP inhibitor for oral cancer therapy in addition
to conventional chemotherapy.

2. Results

2.1. Evaluation of the Cytotoxicity of the Poly(ADP-Ribose) Polymerase (PARP)-1 Inhibitor AZD2281
To investigate the cytotoxic effects of AZD2281 on three cell lines (HSC-2, Ca9-22, and
SAS) derived from oral squamous cell carcinoma, we performed 3-(4,5-dimethyl-2-thiazolyl)-
2,5-diphenyltetrazolium bromide (MTT) assays. The half maximal inhibitory concentration (IC50 ) of
AZD2281 for HSC-2, Ca9-22 and SAS cells after 24 h of treatment was estimated to be 7.2, 15, and
>20 µM, respectively (Figure S1). Next, synergetic effects of AZD2281 and cisplatin were assessed
based on the results of MTT assays. Cell viability was significantly reduced by cisplatin treatment in a
dose-dependent manner. The IC50 of cisplatin was 4.8 µM in HSC-2 cells, 9.1 µM in Ca9-22 cells, and
16.0 µM in SAS cells. With addition of 1 µM AZD2281, the IC50 of cisplatin was 2.4, 4.4, and 8.8 µM in
 Int. J. Mol. Sci. 2016, 17, 272 3 of 19
Int. J. Mol. Sci. 2016, 17, 272 3 of 19

8.8 µM Ca9-22,
HSC-2, in HSC-2,SASCa9-22, SAS cells, respectively
cells, respectively (Figure S2A–C). (Figure S2A–C). Theindex
The combination combination index
(CI) of each cell(CI)
line of
of
each cell line
50% was 0.524offor
50% wascells,
HSC-2 0.5240.549
for HSC-2 cells,cells,
for Ca9-22 0.549andfor0.596
Ca9-22
for cells, and 0.596
SAS cells. forwas
Each CI SASless
cells.
than Each
1.0,
CI was lessthat
indicating thanAZD2281
1.0, indicating that AZD2281
had synergetic effectshad synergetic
with cisplatin.effects with cisplatin.

2.2.
2.2. Relative
Relative Survival
Survival Assay
Assay
In
In survival
survival assays,
assays, combinatorial
combinatorial treatment
treatment with
with 11 oror 33 µM AZD2281
AZD2281 and and cisplatin
cisplatin showed
showed
significantly
significantly higher
higher cytotoxicity
cytotoxicity than
than single
single cisplatin
cisplatin treatment
treatment in in all
all three
three cancer
cancer cell
cell lines
lines (Figure
(Figure
S3A–C).
S3A–C). A A higher
higherdosedoserange
range of of cisplatin
cisplatin waswas
found found
to beto be cytotoxic.
cytotoxic. Concentrations
Concentrations of cisplatin
of cisplatin capable
capable of suppressing
of suppressing cellby
cell survival survival by presence
90% in the 90% in the of 0,presence
1 or 3 µMofAZD2281
0, 1 or were
3 µMapproximately
AZD2281 were 2.1,
approximately
1.5, and 1.2 µM2.1, 1.5, andcells,
in HSC-2 1.2 µM
1.7, in HSC-2
1.4, cells,
and 0.98 µM1.7,
in1.4, and cells,
Ca9-22 0.98 µMandin2.4,
Ca9-22 cells,
1.5 and 1.3and
µM 2.4, 1.5
in SAS
and
cells,1.3 µM in SASThe
respectively. cells, respectively.
combination of 1The
µM combination
cisplatin and of 1 µM1 µM cisplatin
AZD2281 and 1 µM
attenuated AZD2281
growth rates
attenuated growth rates compared with single treatments of either cisplatin
compared with single treatments of either cisplatin or AZD2281 (Figure 1). The results of MTT andor AZD2281 (Figure 1).
The results
survival of MTT
assays and survival
suggested assaysshowed
that AZD2281 suggested that AZD2281
synergetic showed
effects with synergetic
cisplatin effects
in cell lines with
derived
cisplatin
from oralinsquamous
cell lines derived from oral squamous cell carcinoma.
cell carcinoma.

Figure 1.
Figure Attenuatedrelative
1. Attenuated relativecell
cellgrowth
growthbybycombinatorial
combinatorialtreatment
treatmentwithwith11µM cisplatinand
µM cisplatin and 11 µM
µM
AZD2281. Values
AZD2281. Values are
are expressed
expressed as
as the
the mean
mean ˘ SEM.**pp<<0.05;
± SEM. 0.05;****pp<<0.01;
0.01;n.s.,
n.s.,no
nosignificance.
significance.

2.3. Effects
2.3. Effects of
of AZD2281
AZD2281 and
and Cisplatin
Cisplatin on
on Cell
Cell Cycle
Cycle
InIncell
cell cycle analysis,
analysis,cells
cellswerewere treated
treated with with 1 µM cisplatin,
1 µM cisplatin, 1 µM and
1 µM AZD2281 AZD2281 and their
their combination
combination
for 18 h and for 18 h and
allowed allowed
to grow to 24,
for 0, growandfor480,h24,
andand 48 h andAtanalyzed.
analyzed. At 0 G2/M
0 h analysis, h analysis,
arrest G2/M
was
arrest
observedwasinobserved in the
the cisplatin andcisplatin and the group
the combination combination
of HSC-2 group
and of
SAS HSC-2 and and
cell lines, SASboth
cell G2/M
lines, andand
both
S phaseG2/M and
arrest S phase
was observedarrest was
in the observed
cisplatin andinthe
thecombination
cisplatin and the of
group combination group
Ca9-22 cell line. of Ca9-22
Twenty-four
cell
hourline. Twenty-four
after incubation, G2/Mhourarrest
afterwasincubation,
still observedG2/Min thearrest was still observed
same administration group in in allthe
cellsame
lines,
administration
and each cell cycle groupwasinalmost
all cellrecovered
lines, andafter
each48cell cycle was almost
h incubation. recovered
In all cell afterAZD2281
lines, 1 µM 48 h incubation.
showed
In all cell
slight lines,
effects on1 cell
µM cycle
AZD2281 showed
and after 24 hslight effects on
incubation, thecell
cellcycle
cycleandwas after 24 hrecovered
almost incubation, inthe
all cell
cell
cycle was almost
lines (Figure recovered
2A). The in allofcell
population G1lines
phase (Figure 2A). The
in the control population
group of G175.75%,
was 63.95%, phase in andthe control
72.51% in
group
HSC-2,was 63.95%,
Ca9-22, 75.75%,
and SAS cell and
lines,72.51% in HSC-2,
respectively. AfterCa9-22,
cisplatinandandSAS cell lines,drug
combination respectively. After
administration,
cisplatin and combination
each G1 population drug administration,
was dramatically decreased, each G1 population
and recovered after 24was
anddramatically decreased,
48 h incubabation. The
and recovered
population after
of sub G124was
andrelatively
48 h incubabation.
high in HSC-2 Thecell
population
lines (3.53%of sub G1 was
in control relatively
group) comparedhigh in to
HSC-2
anothercell
two lines
cell(3.53% in control
lines (Figure 2B).group) compared to another two cell lines (Figure 2B).
 Int. J. Mol. Sci. 2016, 17, 272 4 of 19
Int. J. Mol. Sci. 2016, 17, 272 4 of 19

Cont.
Figure2.2.Cont.
Figure
Int. J. Mol. Sci. 2016, 17, 272 5 of 19
Int. J. Mol. Sci. 2016, 17, 272 5 of 19

Figure 2. Flow cytometry analysis with propitium iodide after treatment with 1 µM cisplatin, 1 µM
Figure 2. Flow cytometry analysis with propitium iodide after treatment with 1 µM cisplatin, 1 µM
AZD2281, and combinatorial administration. The black arrows indicate G2/M arrest and the red
AZD2281, and combinatorial administration. The black arrows indicate G2/M arrest and the red
arrows indicate S phase arrest (A); and percent distributions of cells in each sub G1, G1, S, and G2/M
arrows indicate S phase arrest (A); and percent distributions of cells in each sub G1, G1, S, and G2/M
phase in each cell lines (B).
phase in each cell lines (B).

2.4. In Vivo Effects of AZD2281 with Cisplatin on Xenografted Tumor Growth

2.4. In Vivo Effects of AZD2281 with Cisplatin on Xenografted Tumor Growth
Xenografted tumors were generated by subcutaneous injection of tumor cells (5 × 106 cells) into
Xenografted tumors were generated by subcutaneous injection of tumor cells (5 ˆ 106 cells) into
the dorsal skin. Only HSC-2 cells could stably generate tumors among the used oral carcinoma cell
the dorsal skin. Only HSC-2 cells could stably generate tumors among the used oral carcinoma cell
lines. Tumor volumes of control group mice increased during the experimental period. The tumor
lines. Tumor volumes of control group mice increased during the experimental period. The tumor
growth of cisplatin and AZD2281 groups significantly decreased compared to the control group, and
growth of cisplatin and AZD2281 groups significantly decreased compared to the control group, and
that of combination group was further decreased (Figure 3A). Cisplatin and AZD2281 groups
that of combination group was further decreased (Figure 3A). Cisplatin and AZD2281 groups showed
showed almost same levels of tumor growth. After five treatments every three days, average tumor
almost same levels of tumor growth. After five treatments every three days, average tumor weights
weights were 0.52, 0.39, 0.38, and 0.27 g in control, cisplatin, AZD2281, and combination groups,
were 0.52, 0.39, 0.38, and 0.27 g in control, cisplatin, AZD2281, and combination groups, respectively
respectively (Figure 3B,C). Thus, AZD2281 treatment (25 mg/kg/day, every three days for five
(Figure 3B,C). Thus, AZD2281 treatment (25 mg/kg/day, every three days for five treatments) with
treatments) with cisplatin was considered to be effective for inhibitory growth of tumors derived
cisplatin was considered to be effective for inhibitory growth of tumors derived from HSC-2 cells
from HSC-2 cells in vivo.
in vivo.
Their body weights were not significantly different both before and after the treatment (Figure S4),
Their body weights were not significantly different both before and after the treatment (Figure S4),
thus confirming that the used treatment protocol was tolerable.
thus confirming that the used treatment protocol was tolerable.
 Int. J. Mol. Sci. 2016, 17, 272 6 of 19
Int. J. Mol. Sci. 2016, 17, 272 6 of 19

Figure
Figure 3.3. (A)
(A)Increases
Increases in tumor
in tumor volumes
volumes duringduring
treatmenttreatment withorcisplatin
with cisplatin AZD2281,ororAZD2281,
combinatorialor
treatment withtreatment
combinatorial both cisplatin
withand AZD2281;
both cisplatin(B) tumor
and weights(B)
AZD2281; justtumor
after sacrifice
weightsinjust
the after
control, cisplatin,
sacrifice in
AZD2281,
the control,and combination
cisplatin, groups;
AZD2281, andand (C) photosgroups;
combination of representative tumors
and (C) photos of in the control, cisplatin,
representative tumors
AZD2281,
in andcisplatin,
the control, combination treatment
AZD2281, and groups. Values
combination are expressed
treatment groups.as Values
the mean SEM. * p as
are˘expressed < 0.05;
the
** p < 0.01.
mean ± SEM. * p < 0.05; ** p < 0.01.

2.5.
2.5. Histopathological
Histopathological Analysis
Analysis Reveals
Reveals that
that the
the Combination
Combination of
of Cisplatin
Cisplatin and AZD2281 Decreases
Proliferation Potential and Increases Necrosis in Tumors
Proliferation Tumors
Microscopic analysis
Microscopic analysis of
of hematoxylin
hematoxylin andand eosin-stained
eosin-stained HSC-2
HSC-2 tumors
tumors ofof the
the three
three treatment
treatment
groups showed hollowed out areas caused by necrosis compared to the
groups showed hollowed out areas caused by necrosis compared to the control control group. The center of
The center of
tumors in the
tumors the combination
combinationgroup
groupshowed
showedlarger
largernecrotic hollowed
necrotic hollowedarea, while
area, tumors
while in cisplatin
tumors and
in cisplatin
AZD2281
and groupsgroups
AZD2281 showedshowed
scatteredscattered
hollowedhollowed
areas (Figure 4). (Figure
areas Coagulative necrosis by chemotherapy
4). Coagulative necrosis by
was also observed
chemotherapy wasoutward from the
also observed center from
outward in thethe
cisplatin
centergroup
in the and partially
cisplatin groupin the
andAZD2281
partially group.
in the
In the combination
AZD2281 group,
group. In the stronger coagulative
combination necrosis
group, stronger was observed
coagulative necrosisinwas
addition to keratinizing
observed in addition
to keratinizing degeneration (Figure S5). Terminal deoxynucleotidyl transferase (TdT) dUTP
(2′-deoxyuridine 5′-triphosphate) nick-end labeling (TUNEL) assays showed that tumors of the
Int. J. Mol. Sci. 2016, 17, 272 7 of 19

Int. J. Mol. Sci. 2016, 17, 272 7 of 19

degeneration (Figure S5). Terminal deoxynucleotidyl transferase (TdT) dUTP (21 -deoxyuridine
5combination
1 -triphosphate) group
nick-endhadlabeling
significantly
(TUNEL) more
assays TUNEL-positive
showed that tumors cells,of the
andcombination
both apoptotic
group had and
pre-apoptotic cells were observed (Figure 5). In the control group,
significantly more TUNEL-positive cells, and both apoptotic and pre-apoptotic cells were observed only a few TUNEL-positive
apoptotic
(Figure 5).cells
In were observed
the control by microscopy,
group, only a fewwhile strong apoptosis
TUNEL-positive was mainly
apoptotic observed
cells were in tumor
observed by
cells in the cisplatin
microscopy, groupapoptosis
while strong and pre-apoptotic
was mainlycellsobserved
were observed
in tumor in small nuclear
cells in stroma group
the cisplatin cells inandthe
AZD2281 group.
pre-apoptotic cellsIn combination
were observed in group,
smallboth
nuclearcellstroma
types showed TUNEL-positivity.
cells in the AZD2281 group. To evaluate the
In combination
therapeutic
group, effect
both cell of the
types combination
showed of cisplatinTo
TUNEL-positivity. and AZD2281,
evaluate tumor cell effect
the therapeutic proliferation ability and
of the combination
microvessel density were also measured. Ki-67 expression was widely
of cisplatin and AZD2281, tumor cell proliferation ability and microvessel density were also measured.observed in tumors of the
control
Ki-67 group, while
expression it was significantly
was widely reduced
observed in tumors ofin
thecisplatin and AZD2281
control group, while it groups to 62.7% (p
was significantly < 0.05)
reduced
and
in 63.9% and
cisplatin (p <AZD2281
0.05), respectively.
groups to 62.7%Consistent
(p < 0.05)with
andthe higher
63.9% (p < sensitivity to the Consistent
0.05), respectively. combination withof
cisplatin and AZD2281, Ki-67 expression was decreased by combinatorial
the higher sensitivity to the combination of cisplatin and AZD2281, Ki-67 expression was decreased treatment to 44.5% of the
control
by group (p <treatment
combinatorial 0.01) (Figure 6). A of
to 44.5% similar tendency
the control was(palso
group observed
< 0.01) (Figure in6).
theAtumor
similarmicrovessel
tendency
density
was alsowhen CD31inexpression
observed the tumorwas evaluated.
microvessel CD31 when
density expression
CD31was strongly was
expression positive in the control
evaluated. CD31
group, however it was significantly reduced in cisplatin and AZD2281 groups
expression was strongly positive in the control group, however it was significantly reduced in cisplatin to 56.8% (p < 0.01) and
64.7%
and (p < 0.01),
AZD2281 groupsrespectively. Moreover,
to 56.8% (p < 0.01) and 64.7% CD31(p < expression was further
0.01), respectively. Moreover, decreased by the
CD31 expression
combinatorial treatment to 24.5% (p < 0.01). Small vessel formation
was further decreased by the combinatorial treatment to 24.5% (p < 0.01). Small vessel formation was hardly observed inwas
the
combination
hardly observed group,
in thewhile it was group,
combination observed in itthe
while wasother threeingroups
observed the other(Figure 7). These
three groups results
(Figure 7).
suggested
These resultsthat decreased
suggested proliferation
that potential, increased
decreased proliferation necrosis
potential, and necrosis
increased apoptosis, andand reduction
apoptosis, and in
vascular formation might be the causes of the slower tumor growth
reduction in vascular formation might be the causes of the slower tumor growth in vivo. in vivo.

Figure 4. Histopathological
Figure Histopathological analysis
analysis of representative xenografted
xenografted tumors in control (A); cisplatin (B);
AZD2281 (C);
AZD2281 (C); and
and combination
combination treatment
treatment(D)
(D)groups.
groups. Scale
Scalebar,
bar,55mm.
mm.
Int. J. Mol. Sci. 2016, 17, 272 8 of 19

Int. J. Mol. Sci. 2016, 17, 272 8 of 19

Figure 5.
Figure 5. (A)
(A) TUNEL
TUNEL staining
staining of xenografted tumors
of xenografted tumors in in control
control (upper
(upper left); cisplatin (upper
left); cisplatin right);
(upper right);
AZD2281 (lower left); and combination (lower right) groups. Open arrows indicate
AZD2281 (lower left); and combination (lower right) groups. Open arrows indicate apoptotic cells, apoptotic cells,
and closed arrows indicate pre-apoptotic cells. Scale bar, 200 µm; (B) TUNEL-positive
and closed arrows indicate pre-apoptotic cells. Scale bar, 200 µm; (B) TUNEL-positive cells (both cells (both
apoptotic and
apoptotic and pre-apoptotic
pre-apoptotic cells)
cells) per
perarea
areain
ineach
eachtreatment
treatmentgroup.
group.****pp<< 0.01.
0.01.
Int. J. Mol. Sci. 2016, 17, 272 9 of 19
Int. J. Mol. Sci. 2016, 17, 272 9 of 19

Figure
Figure 6.
6. (A)
(A)Immunohistochemical
Immunohistochemicalanalysis
analysisofofKi67
Ki67ininxenografted
xenograftedtumors
tumorsofofcontrol
control(upper
(upperleft);
left);
cisplatin (upper right); AZD2281 (lower left); and combination (lower right) groups.
cisplatin (upper right); AZD2281 (lower left); and combination (lower right) groups. Scale bar, Scale
200bar,
µm;
200
(B) µm; (B) Ki67-positive
Ki67-positive cells percells
areaper areatreatment
in each in each treatment
group. **group. ** p < 0.01.
p < 0.01.
 Int. J. Mol. Sci. 2016, 17, 272 10 of 19
Int. J. Mol. Sci. 2016, 17, 272 10 of 19

Figure 7. (A) Immunohistochemical analysis of CD31 in xenografted tumors of the control (upper
Figure 7. (A) Immunohistochemical analysis of CD31 in xenografted tumors of the control (upper
left); cisplatin (upper right); AZD2281 (lower left); and combination (lower right) groups. Scale bar,
left); cisplatin (upper right); AZD2281 (lower left); and combination (lower right) groups. Scale bar,
200
200 µm;(B)
µm; (B)CD31-positive
CD31-positivecells
cellsper
perarea
areain
ineach
eachtreatment
treatment group.
group. **
**pp<<0.01.
0.01.

2.6.
2.6.Results
ResultsofofAnalysis
AnalysisofofWestern
WesternBlotting
Blotting
To assess the mechanism of the synergetic effects of cisplatin and AZD2281, we analyzed the
To assess the mechanism of the synergetic effects of cisplatin and AZD2281, we analyzed the
protein levels of factors related to poly(ADP-ribosyl)ation, vascular endothecial growth factor
protein levels of factors related to poly(ADP-ribosyl)ation, vascular endothecial growth factor (VEGF),
(VEGF), multi-drug resistant gene 1 (MDR1), γ-H2AX, and RAD51 after HSC-2-derived tumors
multi-drug resistant gene 1 (MDR1), γ-H2AX, and RAD51 after HSC-2-derived tumors were resected
were resected on day 18 of medication. Protein levels of PARP-1 and poly(ADP-ribose) (PAR) were
on day 18 of medication. Protein levels of PARP-1 and poly(ADP-ribose) (PAR) were significantly
significantly increased in the combination group compared to the control, and those of VEGF and
increased in the combination group compared to the control, and those of VEGF and MDR1 were
MDR1 were significantly reduced in the combination group, which was consistent with inhibition of
significantly reduced in the combination group, which was consistent with inhibition of tumor growth
tumor growth in vivo. RAD51 expression was significantly reduced in the cisplatin and AZD2281
in vivo. RAD51 expression was significantly reduced in the cisplatin and AZD2281 groups, and further
groups, and further in the combination groups, possibly leading to attenuation of HR. The γ-H2AX
Int. J. Mol. Sci. 2016, 17, 272 11 of 19

Int. J. Mol. Sci. 2016, 17, 272 11 of 19

in the combination groups, possibly leading to attenuation of HR. The γ-H2AX level was significantly
increased
level was only in the AZD2281
significantly increasedgroup, suggesting
only in the significant
the AZD2281 increase of
group, suggesting thedouble-strand breaks in
significant increase of
this condition (Figure
double-strand 8).this condition (Figure 8).
breaks in

Figure 8. (A)
(A) The
The effect of cisplatin and AZD2281 on the protein expression of xenograftedxenografted HSC-2
tumors. Relative band intensity of western blot data was normalized by the expression level of β-actin; β-actin;
The proteins
proteinsanalyzed
analyzedwere
werePARP-1
PARP-1 (poly(ADP-ribose) polymerase-1) (B); poly(ADP-ribose)
(poly(ADP-ribose) polymerase-1) (B); poly(ADP-ribose) (PAR) (PAR)
(C);
(C); vascular
vascular endotherial
endotherial growthgrowth factor (VEGF)
factor (VEGF) (D); multi-drug
(D); multi-drug resistance resistance
gene 1 (MDR1) gene (E);
1 (MDR1)
γ-H2AX (E);
(F);
γ-H2AX
and (F);(G).
RAD51 andValues
RAD51are(G). Values are
expressed as expressed
the mean ˘asSEM.
the mean
* p < ±0.05;
SEM.** p* p< <0.01.
0.05; ** p < 0.01.
Int. J. Mol. Sci. 2016, 17, 272 12 of 19

3. Discussion
In this study, the PARP-1 inhibitor AZD2281 showed in vitro synergetic effects in combination
with cisplatin in three cell lines derived from oral carcinoma, HSC-2, Ca9-22 and SAS, and partly
in vivo synergetic effects in xenografted HSC-2 tumors.
Enhanced anti-tumor effects of PARP inhibitors with cisplatin have been reported, especially in
breast cancer [20–22]. Furthermore, the combination of PARP inhibitors with temozolomide in Ewing’s
sarcoma [23] or camptothecin in childhood neuroblastoma [24] shows stronger synergetic effects
compared to the cisplatin case. In a previous report, cell viability curves were compared between
temozolomide, camptothecin, and cisplatin in DT40 cells (chicken lymphoma cell line) and DU145
cells (prostate cancer cell line), and PARP inhibitor just added its own cytotoxicity [16]. Compared to
previous reports, viability curve of our data showed synergetic effects (Figures S2 and S3). Moreover,
the CI of our data was around 0.55, which was relatively higher than that of DT40 cells and much
lower than that of DU145 cells. Based on our data and previous studies, the CI could be quite different
among cell types.
The synergetic effects of AZD2281 and cisplatin could be related to cell cycle. AZD2281 had been
reported to cause G2/M arrest and decrease DNA repair activity through G2 cell-cycle arrest-like effect
in a dose and p53-dependent manner [25], and cisplatin also causes G2/M arrest [26]. Our data also
showed that the increased G2/M arrest in the combination group could be involved in the synergetic
effects. Moreover, the cell cycle arrest profiles, namely the highest apoptotic sub-G1 population in
the HSC-2 cell line and S-phase arrest in the Ca9-22 cell line, might contribute to the difference in the
sensitivity and cytotoxicity within the three cell lines [27].
In vivo, xenografted HSC-2 tumors also showed the similar synergetic effect after combinatorial
treatment with cisplatin and AZD2281. Although the reduction rate of xenografted tumor volumes was
almost additive (Figure 3A–C), central necrosis, strong TUNEL-positive staining, and reduced numbers
of Ki67-positive cells, which were observed in the combination group, were consistent with the results
obtained from in vitro experiments. The expression level of RAD51 was significantly decreased in the
cisplatin and AZD2281 groups, and even more in the combination group. This could lead to deficient
HR pathway by the combinatorial treatment. Moreover, PARP-1 activation and synthesis of PAR
were much higher in the combination group than other three groups. Consequently, the level of DNA
damage could be highest in the combination group although γ-H2AX expression level was highest in
AZD2281 group.
The mechanism of DNA damage caused by cisplatin is the interaction with DNA to form DNA
adducts, primarily intra-strand crosslink adducts, while the mechanisms of drug resistance could
involve limited DNA damage due to reduction of drug uptake and increase of drug inactivation
and DNA adduct repair. These platinum-DNA adducts’ removal and repair of DNA damage are
mediated mainly by nucleotide excision repair (NER) and HR [28]. PARP-1 is mainly involved in
base excision repair (BER). There are several reports of PARP-1 involvement in NER. For example,
PARP-1 collaborates with DDB2 (damaged DNA-binding protein 2) to increase the efficiency of the
lesion recognition step of global genomic NER [29]. In addition, PARP-1 regulates XPA (xeroderma
pigmentosum complementation group A protein), which is a key protein of NER [30]. Therefore, the
synergetic effects in the combination group might be related to a decrease in the interactions of PARP-1
with DDB2 or XPA, resulting in the increased sensitivity to cisplatin. Of note, our results also showed
that the protein level of MDR1 was significantly decreased in the combination group (Figure 8E).
Therefore, the development of drug resistance might be decreased by the combinatorial treatment with
AZD2281 although the mechanism was not elucidated.
On the other hand, angiogenesis-related factors, such as CD31 and VEGF, were also attenuated
after the combinatorial treatment with AZD2281 and cisplatin, suggesting that AZD2281 is also
involved in inhibition of the angiogenesis pathway. The relationship has been previously reported
between poly(ADP-ribosyl)ation and CD31 and VEGF expression in vascular smooth muscle cells
(VSMCs) [31,32], such as facilitation of DNA repair by prolonged poly(ADP-ribosyl)ation in VSMCs,
Int. J. Mol. Sci. 2016, 17, 272 13 of 19

and the direct effect of PARP inhibitor on tumor vascularization through changes in CD31 expression
levels was reported in in vivo models of anaplastic thyroid carcinoma.
Considering our results and previous studies, synergetic effects of cisplatin and AZD2281 could
be caused by (1) cisplatin effects that cause inter- and intra-strand crosslinks; (2) AZD2281 effects
that attenuate BER; (3) their possible synergetic effects caused through deficient NER and HR; and
(4) a possible direct attenuation of tumor vascularization by AZD2281. Therefore, considering the
therapeutic potential of PARP inhibitors in the treatment of oral cancer, the current study suggested
that combination of cisplatin and AZD2281 is one of the promising candidates for treatment strategy.
Our in vivo experiments were performed using only one cell line, therefore further investigations of the
mechanism responsible for the suppression of HR and NER pathways, or side-effects in combination
with cisplatin and PARP inhibition will be necessary. Meanwhile, it would provide new insights into
cancer therapies.

4. Materials and Methods

All animal experimental procedures were approved by the ethical committee for the guidelines
on animal experiments of Tsurumi University School of Dental Medicine on 3 July 2012 (No. 12040).

4.1. Culture of Cell Lines Derived from Oral Carcinoma

Three cell lines, HSC-2 (human oral squamous cell carcinoma cell line), Ca9-22 (human gingival
carcinoma cell line), and SAS (human tongue squamous cell carcinoma cell line) (all obtained from
RIKEN, Tsukuba, Japan, on 11 October 2012) were used in this study. Growth medium consisted of
10% fetal bovine serum (Biowest, Nuaillé, France), 100 U/mL penicillin, and 100 µg/mL streptomycin
(Sigma-Aldrich, St. Louis, MO, USA) in minimum essential Eagle’s medium (Sigma-Aldrich) for HSC-2
and Ca9-22 cells, and in Dulbecco’s Modified Eagle’s Medium (Sigma-Aldrich) for SAS cells. The cells
were maintained at 37 ˝ C with 5% CO2 . Growth medium was changed every three days. Cells were
passaged at 1:5 after it reached confluence.

4.2. MTT (3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide) Assay

HSC-2, Ca9-22, and SAS oral carcinoma cells were seeded in 24-well plates at a density of
2 ˆ 104 cells/well. After overnight incubation, the culture medium was replaced with fresh medium
containing various concentrations of PARP inhibitor AZD2281 (ChemScene, Monmouth Junction, NJ,
USA) or cisplatin (cis-diammineplatinum(II) dichloride, Sigma-Aldrich). After 24 h of treatment, the
number of viable cells was assessed using an MTT assay as reported previously [33]. Briefly, one tenth
of the fluid volume of 5 mg/mL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
in RPMI-1640 medium (Sigma-Aldrich) was added to each well, followed by incubation for 4 h at
37 ˝ C. After incubation, the medium was carefully removed and an adequate volume of 0.1 N HCl
(Wako Pure Chemical Industries, Ltd., Osaka, Japan) in isopropanol (Wako Pure Chemical Industries,
Ltd.) was added to each well and the resultant formazan crystals was dissolved. Absorbance was
determined at 570 nm by microplate reader (Model 680; Bio-Rad, Hercules, CA, USA) in 96-well assay
plates (Sumilon, Sumitomo Bakelite, Tokyo, Japan). All experiments were performed in triplicate.
IC50 was calculated by Excel software (Microsoft, Redmond, Washington, DC, USA) using the
logarithm function. First, the concentration was plotted on the x-axis, and cell viability was plotted on
the y-axis. Then, using the value of higher and lower sides of the 50% concentration and cell viability,
a linear equation was created as follows:

IC50 “ 10logp A{Bqˆp50´Cq{pD´Cq`logB

where A is the concentration of the higher side of 50% cell viability, B is the concentration of the lower
side of 50% cell viability, C is the cell viability at the concentration of B, and D is cell viability at the
concentration of A.
Int. J. Mol. Sci. 2016, 17, 272 14 of 19

4.3. Survival Assay

2 ˆ 104 cells were seeded in each of 12-well plates and cultured in growth medium with various
concentrations of cisplatin with 0, 1, or 3 µM AZD2281 for 18 h. After washing with phosphate-buffered
saline (PBS), the cells were allowed to grow. Then, cell numbers were counted using a hemocytometer
when cells without AZD2281 reached confluence. Suppression of cell survival by 50% was calculated
using the formula as described above.

4.4. Combination Index Analysis

The results were analyzed using the median effect/CI isobologram equation that is based on
the median effect principle (mass action law) [34,35]. First, the dose effect curves for cisplatin and
AZD2281, and their combination were plotted using the following equation:
ˆ ˙
fa
log “ mlogD ´ mlogDm
fu

where fa is the fraction affected by dose D, the (%) inhibition:

absorbance of MTT assay in drug presence

%inhibition “ ˆ 100
absorbance of MTT assay in drug absence

where fu is the uninhibited fraction (1 ´ fa), D is the administered drug dose (concentration), Dm is the
dose (concentration) required for 50% inhibition (the median effect), and m is defined as the coefficient
of the sigmoidicity of the dose effect curve.
Next, to determine the dose of single drugs (Dx ) or their combination (D1,2)x necessary to achieve
x% inhibition, the median effect relationship was re-expressed as follows:
ˆ ˙ 1{ m
fx
D x “ Dm
1´ fx

where Dx is the dose concentration required for x% inhibition.

Finally, the interaction of the two drugs was assessed by the CI as follows:

D1 combined D2 combined
CI “ `
D1 alone D2 alone

where D1 combined is the dose of cisplatin in combination with AZD2281, D2 combined is the dose of
AZD2281 in combination with cisplatin, D1 alone is the single dose of cisplatin required for Dx with
the combination of cisplatin and AZD2281, and D2 alone is the single dose of AZD2281 required for
Dx with the combination of cisplatin and AZD2281. CI < 1.0 = synergetic, CI = 1.0 = additive, and
CI > 1.0 = antagonistic.

4.5. Flow Cytometry

Cells were seeded in 10 cm-diameter plates at a density of 1 ˆ 105 cells/well and cultured in
growth medium with 1 µM cisplatin or 1 µM AZD2281, and their combination for 18 h. After washing
with PBS, the cells were allowed to grow for 0, 24, and 48 h. Then, each supernatant was collected,
cells were washed with PBS and treated with Accutase (Innovative Cell Technologies, San Diego,
CA, USA) at 37 ˝ C for 10 min. Cells were centrifuged, washed with PBS, and fixed with cold 70%
ethanol for 1.5 h. Subsequently, cells were filtered with a cell strainer (Corning, Corning, NY, USA)
and collected in sample tubes. Cells were then treated with 0.2 mg/mL of RNaseA and 20 µg/mL of
PI (3,8-Diamino-5-(3-(diethylmethylammonio)propyl)-6-phenylphenanthridinium diiodide, Dojindo
Molecular Technologies, Inc., Kumamoto, Japan) in PBS in a dark condition for 30 min. Then, cells
were analyzed by FACS Calibur (Beckton and Dickinson, Franklin Lakes, NJ, USA).
Int. J. Mol. Sci. 2016, 17, 272 15 of 19

4.6. Animal Model

Male Balb/c nu/nu mice (SLC Co., Ltd., Shizuoka, Japan) weighing about 20 g at six-weeks
old each were used in this study. They were maintained under specific pathogen-free conditions
throughout the experiments with constant room temperature, and a 12 h night and day cycle. The
mice were continuously supplied with normal food chow (CE-2; SLC Co., Ltd.) and sterilized water ad
libitum. They were anesthetized and HSC-2, Ca9-22, and SAS cells were injected into their dorsal skin
subcutaneously (5 ˆ 106 cells in 200 µL growth medium per mouse). Within two weeks, tumors usually
enlarged to the diameter of more than 7 mm. Among the three cancer cell lines, only HSC-2 cells
could stably develop tumors. Therefore, in vivo experiments were performed using HSC-2 cell-derived
xenografted tumors.

4.7. Animal Experimental Protocol

Once the tumor diameter had reached 7 mm, the mice were randomly assigned to the following
groups: (a) control (200 µL saline); (b) cisplatin (2 mg/kg per body weight, dissolved in 200 µL
sterilized water); (c) AZD2281 (25 mg/kg per body weight, dissolved in 200 µL sterilized water); or (d)
combination (both cisplatin and AZD2281). The chemicals were administered intraperitoneally every
three days, five times. Although AZD2281 is administered orally in the clinic, intraperitoneal injection
was recommended by the manufacturer because of easier manipulation and the ethical constraints
associated with oral gavage administration to mice. Tumor size and body weight were measured at
the time of administration. The tumor volume was calculated using following equation [36].

Tumor volume “ verticality ˆ width ˆ height ˆ 0.5236

Three days after the last administration, all surviving mice were sacrificed.

4.8. Histopathological Analysis

Mice were treated as described above and then sacrificed. Tumors were removed and immediately
fixed in 10% formalin/PBS (Wako Pure Chemical Industries, Ltd.) for 48 h, followed by immersion in
70% alcohol for 48 h and embedding in paraffin. Tissue sections (4 µm) were mounted on silane-coated
slides (New Silane III, Muto Pure Chemicals Co., Ltd., Tokyo, Japan), deparaffinized with xylene
(Wako Pure Chemical Industries, Ltd.), and rehydrated with graded alcohol solutions (Wako Pure
Chemical Industries, Ltd.). The specimens were pathologically analyzed by hematoxylin and eosin
staining (HE; Merck KGaA, Darmstadt, Germany).

4.9. Terminal Deoxynucleotidyl Transferase (TdT) dUTP Nick-End Labeling (TUNEL) Assay
After deparaffinization with xylene and rehydration with graded alcohol solutions, the sections
were incubated in 20 µg/mL of proteinase K (Boehringer; Mannheim, Germany) dissolved in TBS
(Takara, Ohtsu, Shiga, Japan) for 15 min at room temperature. Then, TUNEL assay was performed
using an ApopTag® Peroxidase in Situ Apoptosis Detection kit (Merck Millipore, Darmstadt, Germany)
according to the manufacturer’s protocol. Apoptotic and necrotic cells were visualized with the Super
Sensitive DAB (3,31 -diaminobenzidine) Substrate Pack (BioGenex, Fremont, CA, USA) according to the
manufacturer’s protocol, followed by counterstaining with hematoxylin and mounting. An apoptotic
cell was defined as a round or oval cell, dense with nuclear staining with DAB, and a pre-apoptotic
cell was defined as a cell with cytoplasmic and nuclear staining with DAB, otherwise normal in
cellular appearance.

4.10. Immunohistochemistry
After deparaffinization with xylene and rehydration with descending concentrations of ethanol,
antigen retrieval was performed using Immunosaver (Nisshin EM, Tokyo, Japan) according to the
Int. J. Mol. Sci. 2016, 17, 272 16 of 19

manufacturer’s protocol. Briefly, sections were incubated in Immunosaver (1:200 dilution in tap water)
for 40 min at 95 ˝ C and then transferred to tap water and incubated for 10 min at room temperature.
Subsequently, endogenous peroxidase was inactivated by treatment with 3% hydrogen peroxide (Wako
Pure Chemical Industries, Ltd.) in methanol (Wako Pure Chemical Industries, Ltd.) for 30 min at room
temperature. After treatment with 20% normal goat serum (Nichirei Corporation, Tokyo, Japan) for
30 min at room temperature, sections were incubated with the primary antibody at 4 ˝ C overnight.
Antibodies used in this study were rabbit polyclonal anti-Ki67 antibody (1:2 dilution, Dako, Troy, MI,
USA) and rabbit polyclonal anti-CD31 antibody (1:50 dilution, Abcam, Cambridge, UK). Antibodies
were diluted with PBS (pH 7.4) containing 1% bovine serum albumin (Sigma-Aldrich) and incubated
at 4 ˝ C. After several PBS washes, bound antibodies were visualized using Histofine Simple Stain
MAX-PO(MULTI) (Nichirei Corporation) and DAB (Vector Laboratories, Inc., Burlingame, CA, USA)
according to the manufacturer’s protocol. The sections were counterstained with hematoxylin and
mounted. As a negative control, sections were processed without exposure to the primary antibody.
The sections were viewed under a BX51 System Microscope (Olympus, Tokyo, Japan). Images were
recorded using a digital microscope camera (DP70; Olympus).

4.11. Western Blotting

A small portion of HSC-2 xenografted tumors in each group were lysed with RIPA buffer (10 mM
Tris-HCl, 1% NP-40, 0.1% SDS, 150 mM NaCl, and 1 mM EDTA) containing protease inhibitor cocktail
(Thermo Fisher Scientific, Waltham, MA, USA). Each tumor was minced and centrifuged at 14,000ˆ g
for 20 min at 4 ˝ C. NuPAGE LDS sample buffer (Invitrogen, Carlsbad, CA, USA) was added to the
supernatant. Subsequently, the samples were heated at 98 ˝ C for 5 min. 80 µg of protein samples
were separated electrophoretically with 8% Bis-Tris gels by the NuPAGE System and subsequently
electroblotted onto a polyvinylidene difluoride membrane using an iBlot Dry Blotting System (all were
from Invitrogen). After blocking with 5% dry skim milk (Wako Pure Chemical Industries, Ltd.) at room
temperature for 30 min, the membranes were treated with primary antibody dissolved in 2.5% dry skim
milk at 4 ˝ C overnight. The primary antibodies used in this study were anti-PARP-1 (1:200 dilution,
Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-PAR (1:1000 dilution, Trevigen, Gaithersburg,
MD, USA), anti-VEGF (1:500 dilution, Bioss Antibodies, Woburn, MA, USA), anti-MDR1 (1:1000
dilution, Bethyl Laboratories, Inc., Montgomery, TX, USA), anti-γ-H2AX (1:250 dilution, Abcam),
and anti-RAD51 (1:500 dilution, GeneTex Inc., Irvine, CA, USA). Then, incubation of membranes
was performed with peroxidase-conjugated secondary antibodies at room temperature for 30 min.
Subsequently, bands were visualized by Amersham ECL Prime Western Blotting Detection Reagent (GE
Healthcare, Buckinghamshire, UK). Then, images were scanned by a C-DiGit Scanner and analyzed by
Image Studio Lite Software (both from Li-COR Biosciences, Lincoln, NE, USA).

4.12. Statistical Analysis

Group comparisons were carried out using an independent t-test. All data were statistically
analyzed by SPSS v.20.0 (IBM Corp., Armonk, NY, USA).

5. Conclusions
In this study, the growth of oral carcinoma cells was attenuated by single treatments with cisplatin
or AZD2281, and further attenuated by combinatorial treatment with combination of cisplatin and
AZD2281 in vitro and in vivo. The in vitro and in vivo results suggest that poly(ADP-ribosyl)ation is
involved in suppression of oral carcinoma growth and the effects are synergetic. Moreover, AZD2281
treatment could decrease the MDR gene expression, suggesting that drug resistance could be prevented
by AZD2281 treatment. Taken together, our results indicate that AZD2281 would be one of a number
of promising molecular-targeted drugs for oral carcinoma therapy.
Int. J. Mol. Sci. 2016, 17, 272 17 of 19

Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/

17/3/272/s1.
Acknowledgments: The study was supported by JSPS (Japan Society for the Promotion of Science) KAKENHI
Grant Numbers 15K11286, 25463157, 24593041, and 21592491.
Author Contributions: Conceived and designed the experiments: Mitsuko Masutani and Hisako Fujihara;
Performed the experiments: Masaaki Yasukawa, Koji Kawaguchi, Ryoko Nakayama, Yuta Kishi,
Nanami Yamamoto and Hiroaki Fujimori; Analyzed the data: Hiroyuki Yamada, Yuta Kishi, Masaaki Yasukawa,
and Hisako Fujihara; Wrote the paper: Masaaki Yasukawa, Yoshiki Hamada, Mitsuko Masutani, and
Hisako Fujihara.
Conflicts of Interest: The authors declare no conflict of interest.

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