Casticin induces apoptosis and G0/G1 cell cycle arrest in gallbladder cancer cells
Song et al. Cancer Cell Int
Casticin induces apoptosis and G0/G1 cell cycle arrest in gallbladder cancer cells
Xiao‑ling Song 0
Zheng Wang 0
Fei Zhang 0
Yi‑jian Zhang 0
Yun‑ping Hu 0
Huai‑feng Li 0
Yuany‑uan Ye 0
Ying‑bin Liu 0
Jun Gu 0
0 Institute of Biliary Tract Disease, Shanghai Jiao Tong University School of Medicine , Shanghai , People's Republic of China
Background: Casticin, the flavonoid extracted from Vitex rotundifolia L, exerts various biological effects, including anti‑ inflammatory and anti‑ cancer activity. The aim of this study is to investigate the effects and mechanisms of casticin in human gallbladder cancer cells. Methods: Human NOZ and SGC996 cells were used to perform the experiments. CCK‑ 8 assay and colony formation assay were performed to evaluate cell viability. Cell cycle analyses and annexin V/PI staining assay for apoptosis were measured using flow cytometry. Western blot analysis was used to evaluate the changes in protein expression, and the effect of casticin treatment in vivo was experimented with xenografted tumors. Results: In this study, we found that casticin significantly inhibited gallbladder cancer cell proliferation in a dose‑ and time‑ dependent manner. Casticin also induced G0/G1 arrest and mitochondrial‑ related apoptosis by upregulating Bax, cleaved caspase‑ 3, cleaved caspase‑ 9 and cleaved poly ADP‑ ribose polymerase expression, and by downregulating Bcl‑ 2 expression. Moreover, casticin induced cycle arrest and apoptosis by upregulating p27 and downregulating cyclinD1/cyclin‑ dependent kinase4 and phosphorylated protein kinase B. In vivo, casticin inhibited tumor growth. Conclusion: Casticin induces G0/G1 arrest and apoptosis in gallbladder cancer, suggesting that casticin might represent a novel and effective agent against gallbladder cancer.
Casticin; Gallbladder cancer; Akt signaling pathway; G0/G1 arrest; Apoptosis
Gallbladder cancer (GBC) is the most common
malignant and fatal tumor of the biliary tract . Diagnostic
and prognostic markers for this malignancy have not
been extensively studied, and, due to lack of
conspicuous symptoms and physical signs, the majority of patients
are diagnosed at an advanced and incurable stage [2, 3].
Moreover, GBC is resistant to chemotherapy or
radiotherapy, surgical resection is the only potentially effective
treatment for GBC [4, 5]. As a result, the overall 5-year
survival rate of GBC is less than 5% [5, 6]. Therefore, the
development of novel and effective agents for GBC
treatment remains a significant challenge.
Flavonoids, which are plentiful in components of human
diets, such as fruits and vegetables, exhibit extensive
biological effects, including anti-cancer, anti-inflammatory,
antioxidant and anti-viral activities . Casticin, the flavonoid
extracted from Vitex rotundifolia L, exerts
anti-inflammatory and anti-cancer activities. Casticin has been
commonly used as an anti-inflammatory agent for thousands
of years in traditional Chinese medicine . In addition,
resent studies has demonstrated that casticin can
alleviate smoke-induced acute lung inflammation . In recent
years, researchers have focused their attention on the
anticancer effects of casticin against lung cancer, cervical
cancer, hepatocellular carcinoma, colon cancer and gastric
cancer [10–14]. However, the effects and mechanisms of
casticin on human GBC cells have yet to be characterized.
© The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,
provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license,
and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/
publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
In this study, we explored the anti-cancer effect of
casticin on GBC cells and investigated the potential
mechanisms mediating these effects. We found that casticin
induced G0/G1 arrest and apoptosis in gallbladder
cancer, suggesting that casticin might represent a novel and
effective agent against gallbladder cancer.
Reagents and drugs
Casticin was obtained from Sigma-Aldrich (St. Louis, MO,
USA) (Fig. 1a), dissolved in dimethyl sulfoxide (DMSO),
and stored at −20 °C. The final DMSO concentration used
was less than 0.1%. A cell counting kit-8 (CCK-8), Hoechst
33342, and Rhodamine 123 were purchased from
SigmaAldrich. Pan-caspase inhibitor (Z-VAD-FMK) and PI3K
inhibitor (LY294002) were obtained from Abcam
(Cambridge, MA, USA). An annexin V/propidium iodide (PI)
apoptosis kit was purchased from Invitrogen (Carlsbad,
CA, USA). TUNEL Apoptosis Assay Kit was purchased
from Beyotime (Shanghai, China). All antibodies were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA,
USA). All cell culture supplies were obtained from
Invitrogen Gibco (Carlsbad, CA, USA).
The human GBC cell lines NOZ and SGC996 were
purchased from the Cell Bank of the Type Culture Collection
of the Chinese Academy of Sciences (Shanghai, China).
NOZ cells were cultured in William’s medium, and
SGC996 cells were cultured in 1640 medium. All media
were supplemented with 100 µg/ml streptomycin and
100 U/ml penicillin (Hyclone, Logan, UT, USA) and 10%
fetal bovine serum (FBS, Gibco). The cells were cultured
at 37 °C in a humidified incubator with 5% CO2.
Cell viability assay
The viability of GBC cells treated with casticin was
evaluated using a CCK-8 assay. Cells were seeded into 96-well
plates at a density of 4000 cells/well and were cultured for
16–24 h. The cells were subsequently treated with
various concentrations of casticin (0, 0.1, 0.5, 1, 4, 7, 10 µM)
for 24, 48 or 72 h. After the treatment, CCK-8 (10 µl) was
added to each well, and the cells were incubated for 3 h
away from light. Absorbance was measured at 450 nm
using a microplate reader (Bio-Tek, Norcross, GA, USA).
Cell viability was calculated using the following formula:
cell viability = (OD of control − OD of treatment)/(OD
of control − OD of blank) * 100%. The assay was repeated
fixed with 10% formalin and stained with 0.1% crystal
violet (Sigma-Aldrich). After washing, the plates were dried up
and the colonies (with more than 50 cells) were observed
under a microscope (Leica, Wetzlar, Germany).
Cell cycle analyses
SGC996 and NOZ cells were treated with casticin (0, 1,
4, 7 µM) for 48 h. The cells were subsequently collected,
washed with phosphate-buffered saline (PBS), and fixed
with 75% ethanol overnight. The cells were then
centrifuged (1500 rpm, 5 min), incubated with 10 mg⁄ml RNase
and 1 mg/ml PI at 37 °C for 30 min away from light.
Ultimately, cell cycle distribution was analyzed by flow
cytometry (FACSCalibur BD, Bedford, MA, USA).
Annexin V/PI staining assay for apoptosis
SGC996 and NOZ cells were treated with casticin (0, 1, 4,
7 µM) for 48 h. Then, the cells were collected and washed
with PBS. After centrifugation (1500 rpm, 5 min), the cells
were combined with 1× Annexin V binding buffer and
then incubated with 5 µl Annexin V and PI at 37 °C for
30 min. Cell apoptosis was measured using flow cytometry.
Hoechst 33342 staining
SGC996 and NOZ cells were treated with casticin (0,
1, 4, 7 µM) for 48 h. The cells were subsequently fixed
with 1 ml methanol/acetic acid (3:1) for 20 min. The
fixed GBC cells were washed with PBS and stained with
5 µg/ml Hoechst 33342 for 15 min at 37 °C. A
fluorescence microscope (Leica, Wetzlar, Germany) was used to
observe the morphological changes.
TUNEL assay was performed on GBC cells and
paraffinembedded tissue sections using the one-step TUNEL
apoptosis assay kit (Beyotime, Shanghai, China)
according to the manufacturer’s instructions. After casticin
treatment, samples were incubated with TUNEL
reaction mixture for 1 h at 37 °C in the dark and then washed
twice in PBS. The condensed or fragmented nuclei of
apoptotic cells were observed using fluorescence
microscopy at 400× magnification.
Mitochondrial membrane potential (ΔΨm) assay
The cells were treated with casticin (0, 1, 4, 7 µM) for
48 h, collected and washed with cold PBS. Then, the
samples were incubated with Rhodamine 123 in a 5% CO2
incubator at 37 °C for 20 min in the dark. Finally, the cells
were analyzed by flow cytometry.
Colony formation assay
The SGC996 and NOZ cells were seeded into 12-well plates
with casticin (0, 1, 4, 7 µM) for 15 days. Then, the cells were
Western blot analysis
Western blot analysis was used to evaluate the changes in
protein expression, as previously described . Proteins
Fig. 1 Casticin inhibits the proliferation and viability of NOZ and SGC996 cells. a The chemical structure of casticin. b, c NOZ, SGC996 and 293T cells
were treated with various concentrations of casticin (0, 0.1, 0.5, 1, 4, 7 µM) for 24, 48 or 72 h. Cell viability was assessed using the CCK‑8 assay. d NOZ
and SGC cells were exposed to 1 µM casticin for 24 h, 48 or 72 h. f, g Casticin suppressed colony formation of NOZ and SGC996 cells. Cells were
exposed to casticin (0, 1, 4, 7 µM) and were allowed to form colonies for 14 days. All data are presented as the means ± standard deviations, and
each experiment was repeated 3 times. Significant differences compared with the control are indicated by *p < 0.05, **p < 0.01, and ***p < 0.001
were separated using 10% sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) and were
then transferred to polyvinylidene difluoride (PVDF)
membranes. The membranes were blocked for 1 h at
37 °C and incubated with primary antibodies against
Bcl-2, Bax, cleaved caspase-9, cleaved caspase-3, cleaved
PARP, CDK4, protein kinase B (AKT), p-AKT, p27,
cyclinD1 and GAPDH overnight at 4 °C. Next, the
membranes were incubated with secondary antibodies for 1 h
at 37 °C. Proteins were observed using a Gel Doc 2000
In vivo tumor xenograft study
Male nude mice (aged 4–6 weeks, weighted 18–22 g)
were purchased from Shanghai SLAC Laboratory Animal
Co Ltd (Shanghai, People’s Republic of China). The
animals were housed at 25 ± 2 °C at a relative humidity of
70 ± 5% under natural light/dark conditions for 1 week
and were allowed free access to food and water. NOZ cells
(at a density of 1 × 106 cells in 0.2 ml) were injected into
the right axilla of each mouse. Twenty-four hours later,
the mice were randomly divided into 3 groups (control,
10, 20 mg/kg; 7 mice per group). Mice in control group
were intraperitoneally injected with (10% DMSO + 90%
PBS). The other groups received administered casticin
(10 or 20 mg/kg) every 2 days for up to 25 days. On day
26, the animals were sacrificed, and the tumors were
dissected and weighed. The animal experiments were
performed in strict accordance with international ethical
guidelines and the National Institutes of Health Guide for
the Care and Use of Laboratory Animals (SYXK
Immunohistochemistry and HE staining
After the mice were sacrificed, tumors from the nude
mice treated with different concentrations of casticin were
resected and immediately fixed in 10% formalin, embedded
in paraffin, cut into 5 mm sections and mounted on slides.
The expression patterns of Ki67, cyclinD1 and p-Akt were
analyzed using immunohistochemical
streptavidin-peroxidase staining (IHC) and hematoxylin and eosin (HE)
staining was used for histopathological examination.
All experiments were performed at least 3 times, and the
results are expressed as the means ± standard deviations
unless otherwise stated. The Student’s t test was used to
compare the differences between the treated groups and
the corresponding control groups. p < 0.05 was
considered statistically significant.
Results and discussion
Casticin inhibits the proliferation and viability of NOZ
and SGC996 cells
Cell proliferation was evaluated using the CCK-8 assay,
which demonstrated that NOZ and SGC996 cell
proliferation rates were significantly inhibited by casticin in a
dose- and time-dependent manner (Fig. 1b–d).
However, casticin treatment did not significantly inhibit 293T
viability (Fig. 1e). The inhibition of casticin-treated GBC
cells was moderate at 48 h, and the IC50 of NOZ and
SGC996 cells was approximately 2 µM at 48 h. Therefore,
we selected 1, 4 and 7 µM as the concentrations to use in
subsequent experiments. The colony formation assay was
used to detect the proliferation of single cell. The NOZ
and SGC996 cells were treated with at various
concentrations (0, 1, 4, 7 µM) for about 2 weeks. As shown in Fig. 1f,
g, the number and size of colonies derived from
casticintreated cells were markedly smaller compared with the
control group. These data demonstrate that casticin can
inhibit the proliferation and viability of GBC cells.
Casticin induces mitochondrial‑dependent apoptosis
in NOZ and SGC996 cells
We investigated the effect of casticin on apoptosis in
GBC cells using flow cytometry and Hoechst 33342
staining. Compared with the control group, the
percentages of casticin-treated cells in the early and late
apoptosis stages were strikingly elevated in a dose-dependent
manner (Fig. 2a–c). In a subsequent experiment, we
treated NOZ and SGC996 cells with various
concentrations (0, 1, 4, 7 µM) for 48 h, and stained the cells with
Hoechst 33342. As shown in Fig. 2d, the casticin-treated
cells exhibited markedly increased chromatin
condensation and fragmentation compared with the control group,
in which cells were round and homogeneously stained.
The result was consistent with the flow cytometry data,
and together, these data indicate that casticin can induce
apoptosis in NOZ and SGC996 cells. TUNEL analysis
also showed more apoptotic cells in casticin-treatment
GBC cells (Fig. 2e).
Mitochondrial damage to cells results in perturbation
of the mitochondrial membrane potential (ΔΨm) .
We evaluated changes in the ΔΨm in NOZ and SGC996
cells using Rhodamine 123 staining, as the decrease in
the intensity of Rhodamine 123 staining reflects
mitochondrial membrane potential and integrity. As shown in
Fig. 3a, b, ΔΨm decreased in a dose-dependent manner,
indicating that casticin induces
Caspases play critical roles in apoptosis initiation and
maintenance [16, 17]. We explored the potential
mechanism of casticin-induced apoptosis using western blot
analysis. As shown in Figs. 3c and 4d, cleaved caspase-3,
-9, -PARP, Bax and p27 were upregulated following
exposure to casticin in a dose-dependent manner, whereas,
Bcl-2, p-Akt and Bcl-2/Bax level significantly decreased
compared with the control group. To confirm the results,
we evaluated cell viability after treatment with casticin
in the presence or absence of Z-VAD-FMK, a caspase
inhibitor. As shown in Fig. 3d, Z-VAD-FMK can abolish
casticin cytotoxicity in GBC cells. Together, these results
indicate that casticin induces mitochondrial-dependent
apoptosis in NOZ and SGC996 cells.
Casticin induces G0/G1 arrest and inhibits proliferation
regulated by an inactive AKT pathway
To determine whether casticin influences cell cycle
progression, we investigated cell cycle distribution by flow
cytometry. The results indicated that the proportions
of G0/G1 cells increased in a dose-dependent
manner in NOZ and SGC996 cells, indicating that casticin
induced G0/G1 arrest (Fig. 4a–c). To further investigate
the effect of casticin on cell cycle progression, we
examined cycle-related protein expression using western blot
analysis. Casticin treatment resulted in decreased
levels of cyclinD1 and CDK4, consistent with a G0/G1 cell
cycle arrest (Fig. 4d). A recent study identified p27 as an
important cyclin-dependent kinase inhibitor that inhibits
Fig. 2 Casticin induces mitochondrial‑ dependent apoptosis in NOZ and SGC996 cells. a–c NOZ and SGC996 cells were treated with casticin (0, 1,
4, 7 µM) for 48 h. The Q3 quadrant (Annexin V−/PI−), Q4 quadrant (Annexin V+/PI−), and Q2 quadrant (Annexin V+/PI+) indicate the percent‑
ages of normal cells, cells in early apoptosis, and cells in late apoptosis, respectively. d NOZ and SGC996 cells were exposed to casticin for 48 h, and
nuclear morphological changes associated with apoptosis were evaluated by Hoechst 33342 staining. d–f Representative images of TUNEL assay
of different casticin‑treatment in GBC cells. All data are presented as the means ± standard deviations and each experiment was repeated 3 times.
Significant differences compared with the control are indicated by *p < 0.05, **p < 0.01, and ***p < 0.001
Fig. 3 Casticin induces mitochondrial‑ dependent apoptosis in NOZ and SGC996 cells. a, b Flow cytometry analysis of the mitochondrial mem‑
brane potential (ΔΨm). NOZ and SGC996 cells were treated with casticin (0, 1, 4, 7 µM) and stained with Rhodamine 123. Cells with high ΔΨm are
marked “survival”, and those with low ΔΨm are marked “apoptosis”. The percentages of cells with low ΔΨm (apoptosis) are shown. c Apoptosis‑
related proteins in NOZ and SGC996 cells were analyzed by western blot. GAPDH was used as a loading control. d After pretreatment with 5 mM
Z‑ VAD‑FMK for 1 h, GBC cells were incubated with 4 µM casticin for 24 h, and cellular viability was determined. All data are presented as the
means ± standard deviations. Significant differences compared with the control were indicated by *p < 0.05, **p < 0.01, and ***p < 0.001
the activation of cyclin D-CDK4 complexes and induces
cell cycle arrest at the G0/G1 and G2/M phases . In
addition, Akt pathway regulates cell cycle progression
and cell proliferation by influencing p27, and the results
presented in Fig. 4d was consistent with these findings.
To determine if casticin-induced proliferation
inhibition was regulated by inhibiting of Akt activity, we
evaluated cell viability after treatment with casticin in the
presence or absence of LY294002, a PI3K/Akt inhibitor.
As shown in Fig. 4e, LY294002 enhanced GBC cells death.
To further confirm the results, we transiently transfected
wild-type Akt and constitutively active Akt into GBC
cells, and then we treated with casticin and evaluated the
viability. As shown in Fig. 4f, overexpression of wild-type
Akt and constitutively active Akt can abolish casticin
cytotoxicity in GBC cells.
Therefore, we conclude that casticin induces G0/G1
arrest via Akt signaling pathway, and that the modulation
of Akt signaling also accounts for the anti-proliferative
effect of casticin.
Casticin inhibits tumor growth in vivo
To evaluate the anti-cancer effect of casticin in vivo,
we injected mice with 10% DMSO + 90% PBS
(control group) or casticin at a concentration of either 10
or 20 mg/kg every 2 days after their inoculation with
NOZ cells. We found that casticin inhibits tumor
growth in a dose-dependent manner (Fig. 5a, b). Based
Fig. 4 Casticin induces G0/G1 arrest by inactiving the AKT pathway. a–c NOZ and SGC996 cells were treated with casticin (0, 1, 4, 7 µM) for 48 h.
The cell cycle distribution was analyzed by flow cytometry. d Expression levels of cyclinD1, CDK4, p27, p‑AKT and AKT were measured using west ‑
ern blot analysis. e After pretreatment with 50 µM LY294002 (Akt inhibitor) for 1 h, GBC cells were incubated with 4 µM casticin for 24 h, and cellular
viability was determined. f Cellular viability was determined after treatment with 4 µM casticin in cells transfected with WT‑Akt, CA‑Akt, or vehicle
plasmid. All data are presented as the means ± standard deviations. Significant differences compared with the control were indicated by *p < 0.05,
**p < 0.01, and ***p < 0.001
on this observation, we performed western blot
analysis, HE and IHC analysis. As shown in Fig. 5c–e, Bcl-2,
cyclinD1, p-AKT and ki-67 expression levels were
strikingly reduced, and Bax expression level was significantly
elevated in casticin-treated groups compared with the
control group. Moreover, tunel analysis showed more
apoptotic cells in casticin-treated groups compared with
the control group (Fig. 5f ). These results are consist with
the in vitro effects of casticin.
GBC is the most common and fatal cancer in biliary
system, and surgical resection is the only effective
treatment option. Thus, it is essential to identify novel
effective treatments for GBC. Traditional Chinese medicine
has been extensively used to treat various diseases for
thousands of years. As an active compound isolated
from Vitex Fructus, casticin can inhibit
proinflammatory cytokines and inflammatory mediators, such as NO
and PGE2, by blocking the activation of NF-ΚB, Akt, and
MAPK signaling . Recent studies have shown that
casticin inhibits proliferation and induces apoptosis in
various cancer cells in vitro. However, there have been no
in vivo tumor xenograft studies evaluating the
anti-cancer effect of casticin. In this study, we investigated NOZ
and SGC996 cell proliferation and viability using CCK-8
analysis and colony formation assays. We found that
casticin can inhibit NOZ and SGC996 cell proliferation,
and casticin cannot significantly inhibit 293T cell
viability. Therefore, we propose that casticin represents a new
and promising therapeutic agent for gallbladder cancer.
Moreover, we evaluated the effect of casticin treatment in
mice with xenografted tumors. Based on the weight and
volume of the tumors, we conclude that casticin exerts
anti-cancer activity in GBC in vivo. In addition, the
expression of related proteins expression using western
blot analysis, HE and IHC staining, the results of which
were in accordance with our in vitro assays.
Fig. 5 Casticin inhibits tumor growth in vivo. a Different concentrations (10% DMSO + 90% PBS, 10 and 20 mg/kg) casticin were injected into
nude mice after inoculated NOZ cells every 2 days. Images of 5 representative mice (n = 7) from each group are presented to show the sizes of the
resulting tumors. b Tumors were excised from the animals and weighed. c–e Bcl‑2, cyclinD1, p ‑AKT and Ki‑67 expression levels were analyzed using
HE, IHC staining and western blot analysis. f Representative images of TUNEL assay of tumor xenografts (original magnifications: ×400). All data are
presented as the means ± standard deviations. Significant differences from the control were indicated by *p < 0.05, **p < 0.01, and ***p < 0.001
We also evaluated the effect of casticin on apoptosis
using flow cytometry, Hoechst 33342 staining and tunel
analysis. Apoptosis is generally characterized as specific
morphological changes, such as cell shrinkage, nuclear
or cytoplasmic fragmentation, chromatin condensation
and the formation of dense bodies that are phagocytosed
by neighboring cells . As shown in Fig. 2d, NOZ and
SGC996 cells treated with casticin exhibited markedly
increased chromatin condensation and fragmentation
compared with the control group cells, which were round
and homogeneously stained. In addition, the proportions
of cells in early and late apoptosis stages in the
casticintreated groups were strikingly elevated in a
Various mechanisms have been suggested to
contribute to the progression of gallbladder cancer, in particular
mutations in components of cell cycle or apoptotic
pathways, and the processes of signal transduction,
angiogenesis, invasion, and metastasis [21–23]. Apoptosis
signaling cascades can be divided into 2 major pathways:
a death-receptor-induced extrinsic pathway and a
mitochondria-apoptosome-mediated intrinsic pathway .
In this study, we found that mitochondrial-dependent
apoptosis was involved in casticin induced apoptosis. Bax
and Bcl-2 are important regulators of the
mitochondriamediated apoptosis pathway, and the balance of these
2 factors is crucial for cell survival and cell death. The
antiapoptotic factor Bcl-2 has been shown to prevent
apoptosis by forming a heterodimer with proapoptotic
factors, such as Bax, resulting in proapoptotic effects
. Activation of Bcl-2 family proteins can induce the
mitochondrial permeabilization, and can induce
caspase-9 activation, which subsequently induces the
cleavage of procaspase-3 . Caspase-3 is a key executioner
caspase that it is capable of cleaving many important
cellular substrates, including PARP . In this study, we
demonstrated that Bax expression significantly increased
and that Bcl-2 expression decreased in response to
casticin treatment both in vitro and in vivo. In addition,
casticin significantly enhanced the enzymatic activity levels
of caspase-3, caspase-9 and PARP (Fig. 3c). Moreover,
ΔΨm decreased in a dose-dependent manner after 48 h
of incubation with casticin. Together, these results
suggest casticin induced apoptosis occurs through the
The induction of cell cycle arrest at a specific
checkpoint and thereby inducing apoptosis is a common
mechanism for the cytotoxic effects of anticancer drugs
. In this study, we investigated cycle distribution
using flow cytometry. The data showed that the
proportion of G0/G1 cells increased in a dose-dependent
manner, indicating that casticin can induce G0/G1 arrest.
Cell cycle progression is highly regulated by a series of
cell cycle checkpoint proteins, such as the cyclins and
CDKs. Among these proteins, cyclinD and E, together
with CDK2, CDK4, or CDK6, play major roles in DNA
replication and mitosis by regulating G0/G1 phase of the
cell cycle . Therefore, we investigated the expression
of cylinD1 and CDK4 in casticin-treated GBC cells and
found that cyclinD1 and CDK4 contributed to G0/G1
The PI3K/AKT pathway is one of the major
signaling pathways involved in the progression of various
tumors and is associated with cancer progression and
invasion . AKT is a key downstream effector of
PI3K and is down-regulated in various cancers,
including osteosarcoma and prostate cancer . Previous
studies have demonstrated that Akt inactivation might
inhibit the expression of proteins associated with events
that mediate the cancer development and progression,
including apoptosis and cell cycle progression [29, 32,
33]. In our study, the results of the western blot
analysis demonstrated that casticin significantly decreased
p-Akt expression and that this effect was accompanied
by an increase in p27. Inactivation of Akt leads to the
increased expression of p27, and decreased expression
of cyclinD1/CDK4 decreased, which contributed to G0/
G1 arrest. Furthermore, Akt inactivation can lead to
upregulation of Bad expression and the downregulation
of Bcl-2 expression, which associated cell apoptosis. In
summary, we suggest that the Akt signaling pathway is
involved in casticin-induced cell apoptosis and cell cycle
Taken together, these findings indicate that the Akt
signaling pathway is involved in casticin-induced cell
apoptosis and cycle arrest. Furthermore, the intrinsic
mitochondrial pathway is involved in casticin-induced
apoptosis. Therefore, we suggest that casticin might be a
novel and effective therapy for GBC.
PARP: poly ADP‑ribose polymerase; CDK4: cyclin‑ dependent kinase4; Akt:
protein kinase B; p‑Akt: phosphorylated protein kinase B; DMSO: dimethyl
sulfoxide; CCK‑8: cell counting kit ‑8; Z ‑ VAD‑FMK: pan‑ caspase inhibitor; FITC:
fluorescein isothiocyanate; PI: propidium iodide; IHC: immunohistochemi‑
cal streptavidin‑peroxidase staining; HE: hematoxylin and eosin; LY294002:
2‑(4‑morpholinyl)‑8‑phenyl‑4H‑1‑benzopyran‑4‑ one; PBS: phosphate buffered
saline; PI3 K: phosphatidylinositol 3‑kinase; z‑ VAD‑fmk: Z ‑ Val‑Ala‑Asp(OMe)‑
CH2F; GAPDH: glyceraldehyde 3‑phosphate dehydrogenase.
XS, YZ designed and conducted the experiments; XW conducted cell viability
and colony formation assay; WZ and ZW conducted annexin V/PI staining
assay for apoptosis and Hoechst 33342 staining; FZ conducted mitochondrial
membrane potential (ΔΨm) assay; JL and JM conducted western blot analysis;
YH, LC and HL conducted in vivo tumour xenograft study and immuno‑
histochemistry. XS and YY analysed the data; XS, YL and JG wrote the main
manuscript text. All authors reviewed the manuscript. All authors read and
approved the final manuscript.
Ethics approval and consent to participate
All animal treatments were carried out in accordance with the National
Institutes of Health Guide for the Care and Use of Laboratory Animals, and
approved by the Institutional Animal Care and Use Committee of Shanghai
Jiaotong University(SYXK [Shanghai] 2013‑0106).
This study was supported by the National Natural Science Foundation of
China (Nos. 81572819, 81172026, 81272402, 81301816, 81172029, 81402403,
81502433 and 31501127), China Postdoctoral Science Foundation (No.
2015M571577), the Program for Changjiang Scholars, the Natural Science
Research Foundation of Shanghai Jiao Tong University School of Medicine (No.
13XJ10037), the Leading Talent program of Shanghai and Specialized Research
Foundation for the Ph.D. Program of Higher Education‑Priority Development
Field (No. 20130073130014), the Interdisciplinary Program of Shanghai Jiao
Tong University (No.14JCRY05), and the Shanghai Rising‑Star Program (No.
1. Lai CH , Lau WY . Gallbladder cancer-a comprehensive review . Surgeon . 2008 ; 6 : 101 - 10 .
2. Cubertafond P , Gainant A , Cucchiaro G . Surgical treatment of 724 carcinoma of the gallbladder . Results of the French surgical association survey . Ann Surg . 1994 ; 219 : 275 - 80 .
3. Nigam Jaya , Chandra Abhijit , et al. Expression of survivin mRNA in gallbladder cancer: a diagnostic and prognostic marker? Tumour Biol . 2014 ; 35 : 9241 - 6 .
4. Rifatbegović Z , Mesić D , Ljuca F , et al. Incidence and surgical treatment of cancer in gallbladder . Med Arhiv . 2007 ; 61 : 30 - 3 .
5. Jiang L , Zhao MN , Liu TY , et al. Bufalin induces cell cycle arrest and apoptosis in gallbladder carcinoma cells . Tumour Biol . 2014 ; 35 : 10931 - 41 .
6. Li M , Zhang Z , Li X , et al. Whole‑ exome and targeted gene sequencing of gallbladder carcinoma identifies recurrent mutations in the ErbB pathway . Nat Genet . 2014 ; 46 : 872 - 6 .
7. Liu HL , Jiang WB , Xie MX . Flavonoids: recent advances as anticancer drugs . Recent Pat Anticancer Drug Discov . 2010 ; 5 : 152 - 64 .
8. Shen JiaK‑un, Huap‑ing Du , Yang Min , et al. Casticin induces leukemic cell death through apoptosis and mitotic catastrophe . Ann Hematol . 2009 ; 88 : 743 - 52 .
9. Lee H , Jung KH , Lee H , Park S , Choi W , Bae H. Casticin , an active compound isolated from Vitex Fructus, ameliorates the cigarette smokeinduced acute lung inflammatory response in a murine model . Int Immunopharmacol . 2015 ; 28 : 1097 - 101 .
10. Koh DJ , Ahn HS , Chung HS , et al. Inhibitory effects of casticin on migration of eosinophil and expression of chemokines and adhesion molecules in A549 lung epithelial cells via NF‑κB inactivation . J Ethnop ‑ harmacol . 2011 ; 136 : 399 - 405 .
11. Chen D , Cao J , Tian L , et al. Induction of apoptosis by casticin in cervical cancer cells through reactive oxygen species‑mediated mitochondrial signaling pathways . Oncol Rep . 2011 ; 26 : 1287 - 94 . doi:10.3892/ or.2011.1367.
12. He L , Yang X , Cao X , et al. Casticin induces growth suppression and cell cycle arrest through activation of FOXO3a in hepatocellular carcinoma . Oncol Rep . 2013 ; 29 : 103 - 8 . doi:10.3892/or. 2012 . 2076 .
13. Tang SY , Zhong MZ , Yuan GJ , et al. Casticin, a flavonoid, potentiates TRAIL‑induced apoptosis through modulation of anti‑apoptotic proteins and death receptor 5 in colon cancer cells . Oncol Rep . 2013 ; 29 : 474 - 80 . doi:10.3892/or.2012.2127.
14. Zhou Y , Tian L , Long L , et al. Casticin potentiates TRAIL‑induced apoptosis of gastric cancer cells through endoplasmic reticulum stress . PLoS ONE . 2013 ; 8 :e58855. doi:10.1371/journal.pone.0058855.
15. Wani ZA , Guru SK , Rao AV , et al. A novel quinazolinone chalcone derivative induces mitochondrial dependent apoptosis and inhibits PI3K/Akt/ mTOR signaling pathway in human colon cancer HCT‑116 cells . Food Chem Toxicol . 2016 ; 87 : 1 - 11 .
16. Nicholson DW , Thornberry NA . Caspases: killer proteases . Trends Biochem Sci . 1997 ; 22 : 299 - 306 .
17. Borner C. The Bcl‑2 protein family: sensors and checkpoints for life ‑ ordeath decisions . Mol Immunol . 2003 ; 39 : 615 - 47 .
18. Chang MY , Shieh DE , Chen CC , et al. Linalool induces cell cycle arrest and apoptosis in leukemia cells and cervical cancer cells through CDKIs . Int J Mol Sci . 2015 ; 16 : 28169 - 79 .
19. Righeschi C , Eichhorn T , Karioti A , et al. Microarray‑based mRNA expression profiling of leukemia cells treated with the flavonoid , casticin. Cancer Genomics Proteomics . 2012 ; 9 : 143 - 51 .
20. Bottone MG , Santin G , Aredia F , et al. Morphological features of organelles during apoptosis: an overview . Cells . 2013 ; 2 : 294 - 305 .
21. Li Z , Chen Y , Wang X , et al. LASP‑1 induces proliferation, metastasis and cell cycle arrest at the G2/M phase in gallbladder cancer by down‑regulating S100P via the PI3K/AKT pathway . Cancer Lett . 2016 ; 372 : 239 - 50 .
22. Cao Y , Liu X , Lu W , et al. Fibronectin promotes cell proliferation and invasion through mTOR signaling pathway activation in gallbladder cancer . Cancer Lett . 2015 ; 360 : 141 - 50 .
23. Shu Y , Weng H , Ye Y , et al. SPOCK1 as a potential cancer prognostic marker promotes the proliferation and metastasis of gallbladder cancer cells by activating the PI3K/AKT pathway . Mol Cancer . 2015 ; 14 : 1 - 14 .
24. Hu W , Kavanagh JJ . Anticancer therapy targeting the apoptotic pathway . Lancet Oncol . 2003 ; 4 : 721 - 9 .
25. Wang J , Lu ML , Dai HL , et al. Esculetin, a coumarin derivative, exerts in vitro and in vivo antiproliferative activity against hepatocellular carcinoma by initiating a mitochondrial‑ dependent apoptosis pathway . Braz J Med Biol Res . 2015 ; 48 : 245 - 53 .
26. Fulda S. Targeting apoptosis for anticancer therapy . Semin Cancer Biol . 2015 ; 31 : 84 - 8 .
27. Bao R , Shu Y , Wu X , et al. Oridonin induces apoptosis and cell cycle arrest of gallbladder cancer cells via the mitochondrial pathway . BMC Cancer . 2014 ; 21 : 217 .
28. Shapiro GI , Harper JW . Anticancer drug targets: cell cycle and checkpoint control . J Clin Investig . 1999 ; 104 : 1645 - 53 .
29. Vermeulen K , Berneman ZN , Van Bockstaele DR . Cell cycle and apoptosis . Cell Prolif . 2003 ; 36 : 165 - 75 .
30. Meng Q , Xia C , Fang J , Rojanasakul Y , Jiang BH . Role of PI3K and AKT specific isoforms in ovarian cancer cell migration, invasion and proliferation through the p70S6K1 pathway . Cell Signal . 2006 ; 18 : 2262 - 71 .
31. Li YJ , Dong BK , Fan M , et al. BTG2 inhibits the proliferation and metastasis of osteosarcoma cells by suppressing the PI3K/AKT pathway . Int J Clin Exp Pathol . 2015 ; 8 : 12410 - 8 .
32. Ma X , Hu Y. Targeting PI3K/Akt/mTOR cascade: the medicinal potential, updated research highlights and challenges ahead . Curr Med Chem . 2013 ; 20 : 2991 - 3010 .
33. Guo SX , Zhou HL , Huang CL , et al. Astaxanthin attenuates early acute kidney injury following severe burns in rats by ameliorating oxidative stress and mitochondrial‑related apoptosis . Mar Drugs . 2015 ; 13 : 2105 - 23 .