Journal of Pharmacological Sciences 127 (2015) 332e338

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Anti-tumor effect of a-pinene on human hepatoma cell lines through

inducing G2/M cell cycle arrest
Weiqiang Chen a, 1, Ying Liu a, 1, Ming Li a, Jianwen Mao a, Lirong Zhang a, Rongbo Huang a,
Xiaobao Jin a, Lianbao Ye b, *
a
School of Basic Courses, Guangdong Pharmaceutical University, Guangzhou, Guangdong, 510006, China
b
Medicinal Chemistry Department, Guangdong Pharmaceutical University, Guangzhou, Guangdong, 510006, China

a r t i c l e i n f o a b s t r a c t s

Article history: Pine needle oil from crude extract of pine needles has been used as an anti-cancer agent in Traditional
Received 2 November 2014 Chinese Medicine. The a-pinene is a natural compound isolated from pine needle oil which has been
Received in revised form shown anti-cancer activity. In previous study, we found that pine needle oil exhibited significant
27 January 2015
inhibitory effect on hepatoma carcinoma BEL-7402 cells. In this study, we investigate the inhibition of a-
Accepted 28 January 2015
pinene on hepatoma carcinoma BEL-7402 cells in vitro and in vivo and further explore the mechanism.
Available online 7 February 2015
The results show that liver cancer cell growth was inhibited obviously with inhibitory rate of 79.3%
in vitro and 69.1% in vivo, Chk1 and Chk2 levels were upregulated, CyclinB, CDC25 and CDK1 levels were
Keywords:
a-Pinene downregulated.
Liver cancer © 2015 Japanese Pharmacological Society. Production and hosting by Elsevier B.V. All rights reserved.
BEL-7402 cells
In vitro and in vivo
Mechanism

1. Introduction a-pinene is a natural compound isolated from pine needle oil which
shows anti-cancer activity. In previous study, we found that pine
Hepatocellular carcinoma is the third leading cause among needle oil exhibited significant inhibitory effect on hepatoma car-
cancer-related death worldwide. It is about 600,000 new cases cinoma BEL-7402 cells in vitro and discussed the mechanism pre-
annually in the world and more than 90% liver cancer is hepato- liminarily (7).
cellular carcinoma (1). Hepatocellular carcinoma is becoming more In this study, we further assessed the inhibitory effect of a-
and more serious to the mankind. The number of deaths owing to pinene on hepatocellular carcinoma BEL-7402 cells in vitro and
hepatocellular carcinoma is increasing dramatically around the in vivo and investigated the molecular mechanism. The results
world each year and the 5-year survival rate is less than 9% (2). The showed that liver cancer cell growth was inhibited obviously, Chk1
clinical treatments for hepatocellular carcinoma are mostly surgical and Chk2 levels were upregulated, CyclinB, CDC25 and CDK1 levels
operation and chemotherapy (3). Therefore, it is necessary to find were downregulated.
novel and effective preventive drugs against human hepatoma.
Pine needles, the leaves of the genus pinus in the family pina- 2. Materials and methods
ceae, contain a substance that can suppress free radicals, regulate
and promote body function, improve health, and reinforce immu- 2.1. Materials
nity (4e6). Pine needle oil from crude extract of pine needles has
been used as an anti-cancer agent in Traditional Chinese Medicine. The a-pinene was isolated from pine needle oil as described
previously (8). The components of pine needle oil were first
analyzed using a gas chromatograph mass spectrometer (GCMS).
* Corresponding author. Medicinal Chemistry Department, Guangdong Pharma- Chromatographic conditions are as follows: HP-5 quartz capillary
ceutical University, Guangzhou Higher Education Mega Center Guangzhou,
column (30 mm
0.25 mm
0.25 mm) was warmed from 50  C to
Guangdong, 510006, China. Tel.: þ86 20 39352139; fax: þ86 20 39352186.
E-mail addresses: yelb7909@163.com, cwq2187@126.com (L. Ye).
160  C at 3  C/min, maintained for 2 min, warmed to 280  C at
Peer review under responsibility of Japanese Pharmacological Society. 10  C/min, and then maintained until the component analysis was
1
These authors contributed equally to this work. finished. Feed volume was 0.2 mL. High purity He (0.99999) was

http://dx.doi.org/10.1016/j.jphs.2015.01.008
1347-8613/© 2015 Japanese Pharmacological Society. Production and hosting by Elsevier B.V. All rights reserved.
 W. Chen et al. / Journal of Pharmacological Sciences 127 (2015) 332e338 333

used as carrier gas with flow rate of 1.0 mL/min. Split ratio was 30:1. group, normal isotonic sodium chloride of 0.2 mL/d as model
For mass spectrum the ion source temperature was 220  C, injec- control, and 5-FU of 0.67 mL/kg as positive control were injected
tion port temperature was 240  C, and electron energy was 70 eV. subcutaneously for 14 ten days every two days. The longest
And diluents at 50, 80 and 100 mmol/L were prepared using tween- diameter (a) and shortest diameter (b) of the tumor were
80 (1%) and serum-free RPMI-1640 medium as described previ- measured using a caliper. Tumor volume was considered equal to
ously (9). (a
b2)/2. Mice with tumor volume larger than 1000 mm3 will be
Methyl thiazolyl tetrazolium (MTT), propidium iodide (PI) and sacrificed. Nude mice were sacrificed two days after the last
RNase A were purchased from Amresco (Solon, OH); RPMI-1640 administration, and tumors were collected and weighed. Calcu-
culture medium and new-born calf serum from Gibco (GrandIs- lated the tumor inhibitory rate according to the following equa-
land, NY); fluorescent quantitative reverse transcription-PCR kit tion. Inhibitory rate was used to evaluate a-pinene's effect against
from Takara (Tokyo, Japan); mouse anti-human cell cycle depend tumors. Inhibitory rate no less than 30% was regarded as valid,
on kinase 1 (CDK1), CyclinB1 and Cdc25C monoclonal antibodies and less than 30% as invalid (11).
were purchased from Millipore (Billerica, MA); actin and rabbit Tumor inhibitory rate ¼ (Average tumor weight of model
anti-human CDK1 p34 (Tyr15) phosphorylated polyclonal anti- contrast group- Average tumor weight of administered group)/
bodies were obtained from Santa Cruz (Santa Cruz Biotechnology Average tumor weight of model contrast group
100%
Inc., Santa Cruz, CA).
2.6. Histology and immunohistology
2.2. Cell culture
Tumor tissues collected from nude mouse xenograft models
Liver cancer cell BEL-7402 was obtained from China Center for were fixed in 4% paraformaldehyde, embedded in paraffin, and cut
Type Culture Collection of Wuhan University and was cultured in into 4 mm sections. After deparaffinization and rehydration, sec-
RPMI-1640 complete culture medium containing 10% new-born tions were processed for H&E staining.
calf serum, 100 U/mL penicillin and 100 mg/mL streptomycin and Immunostaining was performed on deparaffinized tissue sec-
incubated at 37  C in a humidified atmosphere containing 5% CO2. tions. Specimens were treated with 3% H2O2 at room temperature
After several passages, cells in the logarithmic phase were for 30 min, processed for antigen retrieval by microwave, blocked
collected. with normal goat serum for 15 min, and incubated with primary
antibody (1:100) against Chk1, Chk2, CyclinB, CDC25 or CDK1 at
2.3. MTT assay 4  C overnight. For immunochemical staining, sections were incu-
bated at 37  C for 15 min with biotinylated secondary antibody
Liver cancer BEL-7402 cells were maintained in RPMI-1640 (1:100), followed by incubation in horseradish enzyme-labeled
medium, which consists of, 10% fetal bovine serum, 100 U/ml streptomycin avidin at 37  C for 15 min. Sections were then
penicillin and 100 mg/ml streptomycin, at 37  C in a humidified developed in 3, 30 -diaminobenzidine for 30 s. Subsequently, sec-
atmosphere containing 5% CO2. Cell growth in the presence or tions were counterstained with hematoxylin and mounted in
absence of a-pinene was determined using MTT assay (10). Briefly, neutral resin. For immunofluorescence staining, rat anti- or rabbit
rapidly growing cells were harvested, counted and inoculated at anti-monoclonal fluorescent antibodies (1:150) was used as sec-
the concentration of 5
107/mL with 100 mL in each well of 96-well ondary antibodies and samples were incubated with secondary
plate. The cells were then incubated for indicated times in the antibody at 37  C for 60 min. After washing with Tris-buffered sa-
absence or in the presence of different a-pinene. Then 20 mL of line Tween-20, slides were mounted with fluorescent mounting
5 mg/ml MTT was added and incubated in dark at 37  C for 4 h. medium.
After removal of the MTT, cells were treated with 100 uL 5% SDS. All slices were observed under light microscope and were taken
The absorbance was determined at 570 nm using the Automated photographs.
Microplate Reader (Sunrise, Tecan, Switzerland).

2.4. Cell cycle analysis 2.7. Transmission electron microscopy

PI staining was used to analyze the cycling characteristics of cell 2 mL (3
106) of the control and a-pinene-treated cells were
populations. Briefly, logarithmically growing BEL-7402 cells were centrifuged at 2000 r/min for 10 min. Cell pellets were precooled at
treated with 8.4 mM a-pinene for 24, 48 and 72 h. Cells were then 4  C, followed by prefixation in 2% glutaraldehyde and postfixation
harvested and fixed in 70% ethanol at 4  Covernight. Cells were in 1% osmic acid. After dehydration, samples were then embedded
subsequently resuspended in 0.5 mL 50 mg/mL PI staining solution, in epoxy resin and sliced into ultrathin sections. Sections were
kept in the dark at room temperature for 30 min, and analyzed by stained using lead acetate-uranium method. Photoscopy were done
the Coulter Epica XL Flow Cytometer (Beckman, USA). The cell cycle using a transmission electron microscope.
distribution was analyzed with the Muticycle for Windows 32 Bit
USB software. 2.8. Western blot

2.5. Tumor xenograft model and a-pinene treatment Tumor samples were collected (eight days after a-pinene
treatment), washed and homogenized. Equal amounts of protein
30 nude mice aged 5e6 weeks were purchased from Guang- were separated in 10% SDS-PAGE and transferred onto poly-
dong Provincial Experimental Animal Center and were divided vinylidenedifluoride membranes (Millipore, Billerica, MA). Mem-
into three groups (a-pinene-treated group, isotonic sodium branes were probed with primary antibodies (Chk1, Chk2, CyclinB,
chloride group as model control and 5-FU-treated group as pos- CDC25 and CDK1) and then were detected using horseradish
itive control) randomly. 2
106 BEL-7402 cells in logarithmic peroxidase-conjugated goat anti-rabbit IgG (Millpore). Bands were
phase were subcutaneously inoculated in the posterior right hind visualized by enhanced chemiluminescence (ECL Plus, Amersham)
limb of each nude mouse. When tumors reached a diameter of and quantified by AlphaEase FCsoftware (Alpha Innotech, San
3e4 mm. 200 mL of a-pinene (2.67 mL/kg) as a-pinene-treated Leandro, CA).
334 W. Chen et al. / Journal of Pharmacological Sciences 127 (2015) 332e338

progression was investigated. BEL-7402 cells were treated with

either a-pinene or PBS, and cell cycle distribution analysis was then
performed 24 h, 48 h and 72 h. As shown in Fig. 2A, G2/M popu-
lation of a-pinene-treated BEL-7402 cells was increased during the
period of 24e72 h post-treatment, from 7.7% at 0 time point to
23.1% at 72 h post-treatment, while no obvious alteration was
observed in the control cells. This result revealed that a-pinene
treatment caused marked accumulation of a G2/M population in
BEL-7402 cells.

3.3. a-pinene suppresses tumor growth in a xenograft model

Given that a-pinene blocks G2/M progression and cell growth,

we further tested its tumor suppressing ability by using a BEL-7402
xenograft model. Mice bearing tumor xenografts were treated with
Figure 1. Cell growth analysis using MTT assay. BEL-7402 cells were treated with a- a-pinene or 5-FU (positive control) when tumors reached a diam-
pinene with indicated concentration, cells were collected and subject to MTT assay at eter of 3e4 mm. The growth of tumor was then monitored every
indicative time point.
two days. As compared to control group where mice were treated
with isotonic sodium chloride, tumor volume was significantly
inhibited in both a-pinene and 5-FU treated groups (Fig. 2B). No
2.9. Statistical analysis
mice died in the experimental and positive control groups. Two
weeks after treatment, mice were sacrificed and tumors were
The data were analyzed using SPSS 10.0 software, and expressed
measured and weighted. The average tumor size in a-pinene-
as mean ± SD from three separate experiments. The differences
treated group was significantly smaller than that in the control
between groups were analyzed using Student's t test (two-sided).
group (229.21 ± 64.77 mm3 vs. 683.07 ± 42.36 mm3, P < 0.01), and
Differences were considered to be statistically significant at
comparable to 5-FU treated group (323.883 ± 27.8 mm3). Consis-
P < 0.05.
tent with this result, the average tumor weight in a-pinene-treated
group was significantly lighter than the control (0.253 ± 0.093 g vs.
3. Results 0.353 ± 0.032 g, P < 0.05), and similar to that in 5-FU treated group
(0.353 ± 0.032 g). (Table 1). In consistent to this data, histology
3.1. a-pinene inhibits BEL-7402 cell growth examination of xenografts from treated mice showed a lot more
dead cells and slow-growing cells in a-pinene treated and 5-FU
We initiated our test by checking the effect of a-pinene on BEL- treated tumors as compared to the control (Fig. 3). Moreover,
7402 cell growth utilizing MTT assay. BEL-7402 cells were treated apoptosis cell death after a-pinene treatment was also evident from
with a-pinene at the concentrations of 8 mg/L, 2 mg/L, 0.5 mg/L and the scanning electron microscopy result (Fig. 4). Our results suggest
0.125 mg/L, respectively. MTT assay was then applied at 24 h, 48 h that a-pinene significantly suppresses growth of tumor cells in the
and 72 h. As shown in Fig. 1, a-pinene showed inhibitory effect on tumor-bearing mice.
BEL-7402 cell growth in a dose- and time-dependent manner. The
cell growth inhibitory rate was as high as 79.3% (P < 0.05) when a- 3.4. Molecular alteration induced by a-pinene treatment
pinene was applied at the concentration of 8 mg/L for 72 h. The
result suggests that a-pinene can effectively inhibits cell growth. To further understand the mechanism, we explored the mo-
lecular changes that associated with a-pinene in the BEL-7402
3.2. a-pinene blocks BEL-7402 cells in G2/M phase xenograft model. As a-pinene also blocked G2/M transition in our
cell model, a list of G2/M checkpoint proteins were herein analyzed
To better understand the mechanism underlying cell growth by immunohistochemistry. As shown in Fig. 3, the expression of
suppression by a-pinene, the impact of a-pinene on cell cycle Chk1 and Chk2 were increased in a-pinene-treated group and 5-

Figure 2. a-pinene inhibits tumor cell growth in vitro and in vivo. (A) Bel-7402 cells treated with or without a-pinene were collected 48 h after treatment and subject to cell cycle
distribution analysis. Left panel: control group; right panel: a-pinene-treated group. (B) Mice with BEL-7402 xenograft tumors were treated with or without a-pinene. Tumor
growth was monitored and growth curves of tumors are shown.
 W. Chen et al. / Journal of Pharmacological Sciences 127 (2015) 332e338 335

Table 1 (Fig. 6) were therefore performed. Again, we observed upregulated

Inhibitory rate of a-pinene on tumor growth in the nude mice (x±s, n ¼ 8). Chk1 and Chk2 protein level and downregulated Cyclin B, CDC25
Group Tumor Tumor Inhibitory and CDK1 protein level in a-pinene treated cells, as well as 5-FU
volume (mm3) weight (g) rate (%) treated cells which were served as the positive control.
Blank control 683.07 ± 42.36 0.82 ± 0.147 Collectively, our data indicate molecular changes in the G2/M
Positive control 323.883 ± 27.8** 0.353 ± 0.032* 56.911 check point pathway induced by a-pinene, which is associated with
Experimental 229.21 ± 64.77** 0.253 ± 0.093* 69.106 a-pinene induced cell growth inhibition.
**P < 0.01; * < 0.05, vs. blank control group.
4. Discussion
FU-treated group as compared to the control group, while the
expression of Cyclin B, CDC25 and CDK1 decreased. In China, liver cancer is the second most common cause of
To confirm that these molecular changes were induced by cancer morbidity and mortality. About 35, 000 individuals are
a-pinene, immunofluorescence staining (Fig. 5) and western blot diagnosed as liver cancer every year and about 32, 000

Figure 3. Representive photografts of hematoxylineosin staining and immunohistochemistry in BEL-7402 xenografts collected (eight days) after a-pinene treatment. (
200).
336 W. Chen et al. / Journal of Pharmacological Sciences 127 (2015) 332e338

Figure 4. Effect of a-pinene on the protein levels of CDC25, CDK1, CyclinB1, Chk1 and Chk2 in BEL-7402 xenograft tumors collected (eight days/h) after a-pinene treatment.
Immunofluorescence of CDC25C, CDK1, CyclinB1, Chk1 and Chk2 in xenografts. (A) Representative microscopy photografts from immunofluorescence results (
200). (B): Quantified
immunofluorescence signals from (A).

individuals die of liver cancer. Currently, curative measures for regulated by CyclinB1/CDC2 complex in mammalian cells (13,
liver cancer are limited to resection or liver transplantation. 14), and the activity of this complex is regulated by many fac-
However, even though some patients are eligible for operation, tors. For example, dual specificity phosphatase CDC25C activates
the five-year survival rate is only 7% (12). Since Rygarrd firstly CDC2 via upholding phosphorylation on the Thr14 and Tyr15 of
transplanted human tumor cells into nude mice in 1969, nude CDC2 (15). p53 also plays a role on the guardian of G2/M check
mouse xenograft tumor models with human tumor cells are point; p53 activates the transcription of p21 and 14-3-3s (16, 17).
widely used. Thereafter, nude mouse xenograft tumor models While p21 directly suppresses activities of CDC2 (18), and in-
have became an extensively used method in cancer investigation. terferes with PCNA and CDC25 (19), 14-3-3s binds to CyclinB1
Uncontrolled cell growth is one of the characteristics of human and excludes it from the nucleus (20). Moreover, the dissociation
cancers. While cell growth is normally restrained by the control of CDK1-CyclinB1 complexes is also mediated by p53 through
of cell cycle, cell division cycle is under the regulation of the Cdk inducing Gadd45 (Growth arrest and DNA damage inducible
family of serin/threonine kinase and cyclins. G2/M transition is gene) (21, 22).

Figure 5. Representive photografts of BEL-7402 xenografts from each group under an electron microscope (
6000). A: Blank control group; B: 5-FU treated group (positive
control); C: a-pinene treated group.
 W. Chen et al. / Journal of Pharmacological Sciences 127 (2015) 332e338 337

Figure 6. Effect of a-pinene on the protein levels of CDC25, CDK1, CyclinB1, Chk1 and Chk2 in BEL-7402 xenograft tumors collected (eight days) after a-pinene treatment. (A)
Western blot results showing the expression levels of CDC25, CDK1, CyclinB1, Chk1 and Chk2. b-actin was used as the internal control. (B) Summary of a-pinene effects on
expression of CDC25, CDK1, CyclinB1, Chk1 and Chk2. Data were expressed as mean ± SEM of three independent experiments. *P < 0.05 versus control.

In our experiment, we used 5-FU as positive control to test tu- Pharmacological Sciences There are no directly related manuscripts
mor suppressing ability of a-pinene by using a BEL-7402 xenograft or abstracts, published or unpublished, by any authors of this
model because 5-FU has been widely used for cancer patients. The manuscript.
results showed that 5-FU had inhibition on BEL-7402 cell.
Our study found that cell cycle transition from G2 phase to M Acknowledgment
phase was inhibited by a-pinene in BEL-7402 cells. It has also been
reported that inhibition of BEL-7402 cell differentiation by a- This study was supported by Guangdong Natural Science
pinene involves G2/M arrest (23), and this result was confirmed by Foundation (S20130100 16478), Traditional Chinese Medicine City
our experiment. We further found that the Chk2-CDC25 pathway is of South China at Zhongshan, Guangdong Province (2009H016),
involved. The results of immunostaining and western blot revealed Science and Technology Planning Project of Guangdong Province
a reduction of Cyclin B protein level after a-pinene treatment, (2012B031800230), The Guangdong Strategic Emerging Project of
which is associated with G2/M cell cycle arrest. Chk1 and Chk2, the Guangdong Provincial Department of Science and Technology (No.
core of DNA injury check, inhibits CDC2 activity and block mitosis 2012A080800012) and The Joint Natural Sciences Fund of The
by suppressing CDC25 phosphatase activity through phosphoryla- Department of Science and Technology and The First Affiliated
tion. Interestingly, we found Chk1 and Chk2 levels were upregu- Hospital of Guangdong Pharmaceutical University
lated after a-pinene treatment, while CDC25 and CDNK1 levels (No.GYFYLH201317).
were downregulated. Therefore, we presumed that a-pinene
induced cell-cycle arrest is regulated by Chk2 and CDC25C. How- References
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