Osthole inhibits gastric cancer cell proliferation through regulation of PI3K/AKT
Osthole inhibits gastric cancer cell proliferation through regulation of PI3K/AKT
Xiaojun Xu 1 2 3
Xiaoyuan Liu 1 3
Yan Zhang 1 3
0 81671489, Youth project of Wuxi Health Bureau under Grant 2014Z02, Youth Medicine project of Jiangsu province under Grant QNRC2016166 and Doctor Fund of Wuxi Health Bureau under Grant 2014BS01 and Wuxi City Hospital Management Center Pharmacy Program (YGZXY1303). The funders had no role in study design , data collection
1 the National Natural Science Foundation of China under Grant
2 Department of pharmacy, the Second People's Hospital of Wuxi , Wuxi , P.R. China , 2 Department of pharmacy, the Forth People's Hospital of Wuxi , Wuxi , P.R. China , 3 Department of pharmacy, Wuxi M and Child Health Hospital , Wuxi , P.R. China
3 Editor: Salvatore V. Pizzo, Duke University School of Medicine , UNITED STATES
Osthole is an active compound isolated from Chinese herb Cnidium monnieri (L.) Cusson, and had been reported to possess antitumor effect. However, the effect of osthole on the gastric cancer cells has not been investigated. In this study, the effects of osthole on the proliferation of human gastric cancer cells were tested. The data showed that osthole treatment significantly inhibited the proliferation of gastric cancer cells and resulted in the cell cycle arrest at G2/M phase in a dose-dependent manner. Western-blot study showed that the expression of cyclin B1 and cdc2 was markedly reduced by osthole. Moreover, expression of PI3K and pAKT was also significantly suppressed, and the results indicated that the inhibition of pAKT, cyclin B1, and cdc2 levels by osthole was notably enhanced by a PI3K inhibitor. These results demonstrate that osthole could inhibit gastric cancer cells proliferation via induction of cell cycle arrest at G2/M phase by the reduction of PI3K/AKT.
Data Availability Statement; All relevant data are within the paper
Gastric cancer is one of the most aggressive malignancies in the world, especially in China
]. Many progress has been made in the research of gastric cancer, however, surgical
resection has remained the best treatment for gastric cancer still now. Besides, locoregional
recurrence easily occurs even after complete surgical resection . Therefore, it is imperative to
develop novel effective chemotherapeutic drugs for the therapy of gastric cancer.
Osthole, 7-methoxy-8-(3-methyl-2-butenyl)-2H-1-benzopyran-2-one, first isolated from
Cnidium plant, is highly enriched in mature fruit of Cnidium monnieri (Fructus Cnidii) [
Previous experimental data have revealed that osthole exerts a variety of biological and
pharmacological activities including osteogenesis , immunomodulation [
], neuroprote ction
] and antioxidant functions [
], making it a potential functional food and drug candidate. In
recent years, accumulating studies have demonstrated that osthole possesses anti-cancer
property in various kinds of cancers such as ovarian cancer [
], lung cancer [
], sarcoma ,
glioma , leukemia , hepatocellular carcinoma [
], breast cancer  and so on.
However, the influence of osthole on the growth of gastric cancer has not been clarified yet.
Therefore, the purpose of our study was to explore the effect of osthole on the cell growth and cell
and analysis, decision to publish, or preparation of
Competing interests: The authors have declared
that no competing interests exist.
cycle of gastric cancer cells and investigate the possible molecular mechanisms involved, in
order to clarify the biological and therapeutic functions of osthole-treated gastric carcinoma
Materials and methods
Osthole was provided by Green Fount Natural Product Co., Ltd. (Xi'an, China) with a purity
of 98%. RPMI-1640 and trypsin were purchased from Biological Industries (Kibutz Beit
Haemek, Israel). Fetal bovine serum (FBS) was purchased from Solarbio Science&Technology
(Beijing, China). 3-(4, 5-dimethyl thiazol-2yl)-2, 5-diphenyltetrazolium bromide (MTT),
dimethyl sulfoxide (DMSO) and propidium iodide (PI) were obtained from Sigma-Aldrich
(St. Louis, USA). Annexin V-PI apoptosis reagents were from Bytime (Shanghai, China).
Antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Cell culture and cell morphology determination
Human gastric cancer cells SGC-7901 and HGC-27 were obtained from the Shanghai cell
bank of Chinese academy of sciences (Shanghai, China) and kept in our laboratory. The cells
were cultured in RPMI 1640 (Gibco, Invitrogen Corporation, USA) supplemented with 10%
FBS, and maintained in a humidified atmosphere with 5% CO2 at 37ÊC. Osthole was dissolved
in dimethylsulfoxide (DMSO) at a stock solution of 200 mM. Cells were treated with osthole at
final doses of 0±320 μM in culture medium with 10% FBS. After the cells were incubated with
osthole for 48 h, cell morphology was measured using a phase contrast microscope.
Cell viability assay
Cell viability was tested by MTT assay. After treatment with osthole for 48 h, cell viability was
assessed by incubation with 20 μL of 5 mg/ml MTT for 2 h at 37ÊC. Medium with MTT was
removed and 150 μL of DMSO was added. The plate was shaken for 10 min until crystals were
dissolved and measured at 490 nm by an enzyme-linked immunosorbent assay reader
Cell cycle assay
Cell cycle analysis was conducted by flow cytometry as described previously [
]. The cells
were treated with various doses of osthole for 48 h, harvested and fixed in 70% ethanol at 4ÊC.
After 48 h, the cells were rinsed with PBS, incubated with RNase (50 μg/ml), and stained with
PI (100 μg/ml) in the dark for 30 min. The cell phase distribution was then tested using a
FACScan flow cytometer (Becton Dickinson, San Jose, CA).
Cell apoptosis assay
Cell apoptosis detection was performed using Annexin V-PI staining and flow cytometry
analysis. The cells were treated with various doses of osthole for 48 h, harvested and re-suspended
in binding buffer. Then the cells were incubated with Annexin V solution, and stained with PI
solution in the dark for 15 min. The cell apoptosis was then detected using a FACScan flow
cytometer (Becton Dickinson, San Jose, CA).
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Western blot assay
The cells were harvested, wash with PBS and lysed (lysis buffer: 10 mmol/L Tris-HCl (pH
7.4), 150 mmol/L NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 5 mmol/L
edetic acid, 1 mmol/L phenylmethysulfonyl fluoride (PMSF), 0.28 kU/L aprotinin, 50
mg/L leupeptin, 1 mmol/L benzamidine, and 7 mg/L pepstatin A). The protein
concentration was measured using a bicinchoninic acid (BCA) kit. Proteins were separated by
SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were
blocked with 5% skimmed milk in Tris buffered saline (TBS) containing 0.1% Tween 20,
and incubated with primary antibodies overnight at 4ÊC. The membranes were washed
and incubated with fluorescent secondary antibody for 1 h. The ratio of the protein
interested was subjected to GAPDH and was densitometrically analyzed by Odyssey
infrared imaging system [
Data were expressed as mean ± SD. For statistical analysis, one-way analysis of variance
(ANOVA) was performed for comparisons among groups using SPSS 13.0 software. A
twoside P value <0.05 was considered statistically significant.
Osthole inhibits the cell proliferation of gastric cancer cells
To assess the anti-gastric cancer activity of osthole (Fig 1A), HGC-27 and SGC-7901 cells
were treated with the indicated concentrations of osthole for 48 h, and the cell viability
was examined using MTT assay. As shown in Fig 1B, osthole inhibited the cell viability of
HGC27 and SGC-7901 cells in a dosage-dependent fashion. As the microscopy images in
Fig 1C shown, after treated with 20, 40 or 80 μmol/l osthole for 48 h, the HGC27 and
SGC-7901 cells exhibited obvious morphological features, such as shrinkage and
distortion; besides, osthole treated cells became rounded, which was consistent with the MTT
Osthole induces cell cycle arrest at G2/M phase in gastric cancer cells
To determine the possible mechanisms of osthole-mediated cell growth inhibition in
HGC27 and SGC-7901 cells, cell cycle distribution was analyzed. HGC27 and SGC-7901
cells were treated with 20, 40 or 80 μmol/l osthole for 48 h and then the cell cycle phase was
analyzed using PI staining and flow cytometry. As shown in Fig 2A and 2C, upon
incubation with 20, 40 or 80 μmol/l osthole, the percentage of cells in G2/M phase were 24.2%,
29.7% and 32.2% after 48 h in HGC-27 cells, respectively, while the G0/G1 phase
distribution was decreased between 61.0%, 52.8% and 50.8% when treated with 20, 40 or 80 μmol/l
osthole, respectively. The similar cell cycle distribution pattern was also found in SGC-7901
cells in a concentration-dependent manner (Fig 2B and 2D). To further confirm whether
osthole induced cell growth inhibition was associated with cell apoptosis, Annexin V-PI
staining was conducted. As shown in Fig 2E and 2F, osthole treatment did not obviously
induce gastric cell apoptosis at doses of 20, 40 or 80 μmol/l in HGC-27 cells, implying that
osthole induced gastric cancer cell growth inhibition was not due to cell apoptosis.
Collectively, these data revealed that osthole inhibits gastric cancer cell growth by induction of cell
cycle arrest at G2/M phase.
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Fig 1. Osthole inhibits the cell growth of gastric cancer cells. (A) Chemical structure of osthole. (B) HGC-27 and
SGC-7901 cells were treated with the indicated concentrations of osthole for 48 h, and the cell viability was measured
by MTT assay. (C) HGC-27 and SGC-7901 cells were treated with or without osthole for 48 h and the cell morphology
was determined under light microscopy (magnification, ×400). P<0.01 vs. control.
Osthole represses PI3K-AKT signaling in gastric cancer cells
The PI3K/Akt is one of the most important signaling pathways in controlling cell proliferation,
cell cycle and apoptosis and plays a pivotal role in the development, progression and metastasis
of various cancer cells [
]. In order to verify whether osthole mediated gastric cancer cells
inhibition at G2/M phase was associated with PI3K/Akt signaling, we explored the expression
of PI3K, pAkt and Akt after incubation with the different dosages of osthole for 48 h. As
shown in Fig 4A and 4B, the levels of PI3K and its downstream target pAkt decreased
significantly in a concentration-dependent manner exposure to osthole, while the total level of Akt
was slightly changed in response to osthole, suggesting that PI3K/Akt signaling plays an
important role in osthole mediated cell cycle arrest in gastric cancer cells.
To further confirm whether PI3K/Akt signaling was involved in osthole induced gastric
cancer cell cycle arrest, we using a PI3K specific inhibitor, LY294002, in the following study.
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Fig 2. Osthole induces cell cycle arrest in gastric cancer cells. HGC-27 (A) and SGC-7901 (B) cells were treated with
or without osthole for 48 h and the cell cycle proportions were determined by PI staining and flow cytometry.
Quantitative data indicate cell cycle distribution of osthole-treated HGC-27 (C) and SGC-7901 (D) cells. HGC-27 cells
were treated with or without osthole for 48 h and the cell apoptosis were assessed by Annexin V-PI staining (E).
Quantitative data indicates cell apoptosis of osthole-treated HGC-27 cells (F). P<0.01 vs. control.
LY294002 pretreatment notably decreased the phosphorylation level of Akt compared with the
osthole group in gastric cancer cells, while the total Akt level was unchanged (Fig 4C and 4D).
Moreover, our western-blot data also showed that LY294002 pretreatment restored osthole
induced down-regulation of cyclin B1 and cdc2 in human gastric cancer cells (Fig 4C, 4E and
4F). These results suggested that osthole induced cell cycle arrest at G2/M phase of gastric
cancer cells by the modulation of the PI3K/Akt signaling.
It has been demonstrated that osthole, a coumarin derivative, possesses various
pharmacological properties including immunomodulation, osteogenesis, neuroprotection and antioxidation
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Fig 3. Osthole suppresses the expression of cyclinB1 and cdc2 proteins in gastric cancer cells. HGC-27 (A) and SGC-7901
(B) cells were treated with or without osthole for 48 h and subjected to sodium dodecyl sulfate-polyacrylamide gel
electrophoresis and analyzed by western blot assay. The expression of cyclinB1 and cdc2 was measured by western blot assay.
Densitometric analyses of cyclinB1 and cdc2 proteins were expressed as the mean ± SD from three independent experiments
(C: HGC-27, D: SGC-7901). P<0.05, P<0.01 vs. control.
and antitumor activities [
]. In terms of its anti-cancer property, osthole has been
revealed to induce cell proliferation inhibition, apoptosis, cell cycle arrest and cell migration
suppression in various tumor cells [
]. Nevertheless, no detailed studies have
been demonstrated on its effect on gastric caricinoma cells still now. Our study aimed to
elucidate the anti-gastric caricinoma activity of osthole and its underlying molecular mechanisms,
and our data showed that osthole effectively inhibited cell growth and stimulated cell cycle
arrest at G2/M phase in both gastric cancer HGC27 and SGC-7901 cells.
One of the major characteristics is the loss of control on cell proliferation, which develops
partly due to the excessive regulation of cellular growth. The process of cell cycle depends on
the strict control of cell cycle at different levels, and the core of these regulatory factors is
consists of cyclins-dependent kinases (CDKs), cyclin and CDK inhibitors (CDKIs) [
important regulators of the cell cycle machinery, CDKs are activated in normal cells and promote
cell cycle progression from one to the next. Nevertheless, there are many changes in tumor
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Fig 4. Osthole represses PI3K-AKT signaling in gastric cancer cells. HGC-27 cells were treated with or without osthole for 48 h and
the cell lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analyzed by western blot assay (A).
The expression of PI3K, pAKT and AKT proteins was determined by western blot assay. Densitometric analyses of PI3K, pAKT and
AKT proteins were expressed as the mean ± SD from three independent experiments (B). HGC-27 cells were pretreated with PI3K
specific inhibitor LY294002 for 2 h and followed by the indicated concentration of osthole, the cell lysates were subjected to sodium
dodecyl sulfate-polyacrylamide gel electrophoresis and analyzed by western blot assay. The expression of pAKT, AKT, cyclin B1, and
cdc2 proteins was determined by western blot assay (C). Densitometric analyses of these proteins were expressed as the mean ± SD
from three independent experiments (D: pAKT, AKT; E: cyclin B1; F: cdc2). P<0.01 vs. control; ## P<0.01 vs osthole group.
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cells, including CDK genes mutation, cyclins amplification and inactivation of CDKIs, which
causes the dysfunction of CDK activity, mutation of cell cycle checkpoint, abnormal
amplification of cell growth signals, resistance to cell apoptosis and so on [
]. Thus, searching for
effective agents of cell cycle arrest induction has become one of the hotspots of cancer research. We
evaluated the anti-gastric cancer effect of osthole on gastric cancer HGC27 and SGC-7901 cells
using a MTT assay, and the results revealed that osthole effectively inhibited gastric cancer cell
proliferation in a dosage-dependent pattern. Flow cytometric analysis indicated that osthole
induced G2/M phase arrest in a dosage-dependent pattern, which might be associated with the
down-regulation of cyclin B1 and cdc2. These data provided strong evidence in support of the
notion that osthole has potent activity of anti-gastric carcinoma via cell cycle arrest and
suppression of cyclin B1 and cdc2 in vitro.
Many documents have demonstrated that PI3K/Akt signaling pathway play an important
role in the procession of cellular survival. PI3K/Akt signaling activates and promotes the
phosphorylation of Akt cascade, which prevents oxidative damage. The regulation of osthole on
PI3K/Akt signaling has been found in several cancers, including sarcoma , glioma [
and lung cancer [
]. However, whether osthole suppresses gastric cancer cell growth was
associated with PI3K/Akt signaling is not reported yet. Therefore, we evaluated the effect of
osthole on PI3K/Akt signaling in gastric cancer cells. Our data showed that osthole effectively
inhibited the expression of PI3K and p-Akt in gastric cancer cells, whereas the expression of
total Akt is not obviously affected by osthole. Moreover, LY294002, a specific inhibitor of
PI3K, has been used to further confirm the role of PI3K/Akt signaling in osthole mediated
gastric cancer cells growth inhibition. The results showed that LY294002 pretreatment notably
decreased the phosphorylation level of Akt compared with the osthole group in gastric cancer
cells, and restored osthole induced down-regulation of cyclin B1 and cdc2 in human gastric
cancer cells, implying that osthole may suppress gastric cancer cell cycle arrest via
down-regulating PI3K/Akt signaling.
In summary, our data demonstrated that osthole exhibited a potential anti-gastric cancer
property by inducing cell cycle arrest at G2/M phase. Moreover, our study also showed that
PI3K/Akt signaling plays a key role in osthole mediated cell cycle arrest. Osthole could be a
potential drug candidate for the treating of gastric cancer.
This work was supported by grants from the National Natural Science Foundation of China
under Grant #81401222 and #81671489, Youth project of Wuxi Health Bureau under Grant
2014Z02, Youth Medicine project of Jiangsu province under Grant QNRC2016166 and Doctor
Fund of Wuxi Health Bureau under Grant 2014BS01.
Conceptualization: Yan Zhang.
Data curation: Xiaojun Xu, Yan Zhang.
Formal analysis: Xiaoyuan Liu, Yan Zhang.
Funding acquisition: Yan Zhang.
Methodology: Xiaojun Xu, Yan Zhang.
Project administration: Yan Zhang.
Resources: Yan Zhang.
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Software: Xiaoyuan Liu, Yan Zhang.
Supervision: Xiaojun Xu, Yan Zhang.
Validation: Yan Zhang.
Visualization: Xiaoyuan Liu, Yan Zhang.
Writing ± original draft: Xiaoyuan Liu.
Writing ± review & editing: Xiaojun Xu, Yan Zhang.
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1. Venerito M , Link A , Rokkas T , Malfertheiner P . Gastric cancerÐclinical and epidemiological aspects . Helicobacter . 2016 ; 21 Suppl 1 : 39 ± 44 .
2. Gao P , Tsai C , Yang Y , Xu Y , Zhang C , Zhang C , et al. Intraoperative radiotherapy in gastric and esophageal cancer: meta-analysis of long-term outcomes and complications . Minerva Med . 2017 ; 108 : 74 ± 83 . https://doi.org/10.23736/S0026-4806. 16 . 04628 -0 PMID: 27701375
3. Mizrak KD , Harada K , Shimodaira Y , Amlashi FG , Lin Q , Ajani JA . Advanced gastric adenocarcinoma: optimizing therapy options . Expert Rev Clin Pharmacol . 2017 ; 10 : 263 ± 271 . https://doi.org/10.1080/ 17512433. 2017 .1279969 PMID: 28094573
4. Chen R , Xue J , Xie M. Osthole regulates TGF-beta1 and MMP-2/9 expressions via activation of PPARalpha/gamma in cultured mouse cardiac fibroblasts stimulated with angiotensin II . J Pharm Pharm Sci . 2013 ; 16 : 732 ± 741 . PMID: 24393555
5. Zhang ZR , Leung WN , Cheung HY , Chan CW . Osthole: A Review on Its Bioactivities, Pharmacological Properties, and Potential as Alternative Medicine . Evid Based Complement Alternat Med . 2015 ; 2015 : 919616. https://doi.org/10.1155/ 2015 /919616 PMID: 26246843
6. Zhai YK , Pan YL , Niu YB , Li CR , Wu XL , Fan WT , et al. The importance of the prenyl group in the activities of osthole in enhancing bone formation and inhibiting bone resorption in vitro . Int J Endocrinol . 2014 ; 2014 : 921954. https://doi.org/10.1155/ 2014 /921954 PMID: 25147567
7. Yan YH , Li SH , Li HY , Lin Y , Yang JX . Osthole Protects Bone Marrow-Derived Neural Stem Cells from Oxidative Damage through PI3K/Akt- 1 Pathway. Neurochem Res . 2017 ; 42 : 398 ± 405 . https://doi.org/ 10.1007/s11064-016-2082-y PMID: 27734182
8. Li K , Ding D , Zhang M. Neuroprotection of Osthole against Cerebral Ischemia/Reperfusion Injury through an Anti-apoptotic Pathway in Rats . Biol Pharm Bull . 2016 ; 39 : 336 ± 342 . https://doi.org/10. 1248/bpb.b15-00699 PMID: 26934926
9. Yang SM , Chan YL , Hua KF , Chang JM , Chen HL , Tsai YJ , et al. Osthole improves an accelerated focal segmental glomerulosclerosis model in the early stage by activating the Nrf2 antioxidant pathway and subsequently inhibiting NF-kappaB-mediated COX-2 expression and apoptosis . Free Radic Biol Med . 2014 ; 73 : 260 ± 269 . https://doi.org/10.1016/j.freeradbiomed. 2014 . 05 .009 PMID: 24858719
10. Jiang G , Liu J , Ren B , Tang Y , Owusu L , Li M , et al. Anti-tumor effects of osthole on ovarian cancer cells in vitro . J Ethnopharmacol . 2016 ; 193 : 368 ± 376 . https://doi.org/10.1016/j.jep. 2016 . 08 .045 PMID: 27566206
11. Xu XM , Zhang ML , Zhang Y , Zhao L . Osthole induces lung cancer cell apoptosis through inhibition of inhibitor of apoptosis family proteins . Oncol Lett . 2016 ; 12 : 3779 ± 3784 . https://doi.org/10.3892/ol. 2016 . 5170 PMID: 27895730
12. Feng H , Lu JJ , Wang Y , Pei L , Chen X . Osthole inhibited TGF beta-induced epithelial-mesenchymal transition (EMT) by suppressing NF-kappaB mediated Snail activation in lung cancer A549 cells . Cell Adh Migr . 2017 : 1 ± 12 .
Wang L , Yang L , Lu Y , Chen Y , Liu T , Peng Y , et al. Osthole Induces Cell Cycle Arrest and Inhibits Migration and Invasion via PTEN/Akt Pathways in Osteosarcoma . Cell Physiol Biochem . 2016 ; 38 : 2173 ± 2182 . https://doi.org/10.1159/000445573 PMID: 27185245
14. Ding D , Wei S , Song Y , Li L , Du G , Zhan H , et al. Osthole exhibits anti-cancer property in rat glioma cells through inhibiting PI3K/Akt and MAPK signaling pathways . Cell Physiol Biochem . 2013 ; 32 : 1751 ± 1760 . https://doi.org/10.1159/000356609 PMID: 24356539
Wang H , Jia XH , Chen JR , Wang JY , Li YJ . Osthole shows the potential to overcome P-glycoproteinmediated multidrug resistance in human myelogenous leukemia K562/ADM cells by inhibiting the PI3K/Akt signaling pathway . Oncol Rep . 2016 ; 35 : 3659 ± 3668 . https://doi.org/10.3892/or. 2016 .4730 PMID: 27109742
16. Zhang L , Jiang G , Yao F , Liang G , Wang F , Xu H , et al. Osthole promotes anti-tumor immune responses in tumor-bearing mice with hepatocellular carcinoma . Immunopharmacol Immunotoxicol . 2015 ; 37 : 301 ± 307 . https://doi.org/10.3109/08923973. 2015 .1035391 PMID: 25975579
Wang L , Peng Y , Shi K , Wang H , Lu J , Li Y , et al. Osthole inhibits proliferation of human breast cancer cells by inducing cell cycle arrest and apoptosis . J Biomed Res . 2015 ; 29 : 132 ± 138 . https://doi.org/10. 7555/JBR.27.20120115 PMID: 25859268
18. Yuan L , Zhang Y , Xia J , Liu B , Zhang Q , Liu J , et al. Resveratrol induces cell cycle arrest via a p53-independent pathway in A549 cells . Mol Med Rep . 2015 ; 11 : 2459 ± 2464 . https://doi.org/10.3892/mmr. 2014 .3100 PMID: 25515619
19. Zhao L , Miao HC , Li WJ , Sun Y , Huang SL , Li ZY , et al. LW -213 induces G2/M cell cycle arrest through AKT/GSK3beta/beta-catenin signaling pathway in human breast cancer cells . Mol Carcinog . 2016 ; 55 : 778 ± 792 . https://doi.org/10.1002/mc.22321 PMID: 25945460
20. Li L , Lu N , Dai Q , Wei L , Zhao Q , Li Z , et al. GL-V9, a newly synthetic flavonoid derivative, induces mitochondrial-mediated apoptosis and G2/M cell cycle arrest in human hepatocellular carcinoma HepG2 cells . Eur J Pharmacol . 2011 ; 670 : 13 ± 21 . https://doi.org/10.1016/j.ejphar. 2011 . 08 .054 PMID: 21944925
21. Morgensztern D , McLeod HL . PI3K/Akt/mTOR pathway as a target for cancer therapy . Anticancer Drugs . 2005 ; 16 : 797 ± 803 . PMID: 16096426
22. Yoon MK , Mitrea DM , Ou L , Kriwacki RW . Cell cycle regulation by the intrinsically disordered proteins p21 and p27 . Biochem Soc Trans . 2012 ; 40 : 981 ± 988 . https://doi.org/10.1042/BST20120092 PMID: 22988851
23. Muller S , Almouzni G . Chromatin dynamics during the cell cycle at centromeres . Nat Rev Genet . 2017 ; 18 : 192 ± 208 . https://doi.org/10.1038/nrg. 2016 .157 PMID: 28138144
24. Xu X , Zhang Y , Qu D , Jiang T , Li S . Osthole induces G2/M arrest and apoptosis in lung cancer A549 cells by modulating PI3K/Akt pathway . J Exp Clin Cancer Res . 2011 ; 30 : 33 . https://doi.org/10.1186/ 1756 -9966-30-33 PMID: 21447176