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In Vivo and In Vitro Suppression of Hepatocellular Carcinoma by EF24, a Curcumin Analog

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The synthetic compound 3,5-bis(2-flurobenzylidene)piperidin-4-one (EF24) is a potent analog of curcumin that exhibits enhanced biological activity and bioavailability without increasing toxicity. EF24 exerts antitumor activity by arresting the cell cycle and inducing apoptosis, suppressing many types of cancer cells in vitro. The antiproliferative and antiangiogenic properties of EF24 provide theoretical support for its development and application to liver cancers. We investigated the in vitro and in vivo activities of EF24 on liver cancer to better understand its therapeutic effects and mechanisms. EF24 induced significant apoptosis and G2/M-phase cell cycle arrest in mouse liver cancer cell lines, Hepa1-6 and H22. The expression levels of G2/M cell cycle regulating factors, cyclin B1 and Cdc2, were significantly decreased, pp53, p53, and p21 were significantly increased in EF24-treated cells. In addition, EF24 treatment significantly reduced Bcl-2 concomitant with an increase in Bax, enhanced the release of cytochrome c from the mitochondria into the cytosol, resulting in an upregulation of cleaved-caspase-3, which promoted poly (ADP-ribose) polymerase cleavage. EF24-treated cells also displayed decreases in phosphorylated Akt, phosphorylated extracellular signal-regulated kinase and vascular endothelial growth factor. Our in vitro protein expression data were confirmed in vivo using a subcutaneous hepatocellular carcinoma (HCC) tumor model. This mouse HCC model confirmed that total body weight was unchanged following EF24 treatment, although tumor weight was significantly decreased. Using an orthotopic HCC model, EF24 significantly reduced the liver/body weight ratio and relative tumor areas compared to the control group. In situ detection of apoptotic cells and quantification of Ki-67, a biomarker of cell proliferation, all indicated significant tumor suppression with EF24 treatment. These results suggest that EF24 exhibits anti-tumor activity on liver cancer cells via mitochondria-dependent apoptosis and inducing cell cycle arrest coupled with antiangiogenesis. The demonstrated activities of EF24 support its further evaluation as a treatment for human liver cancers.
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In Vivo and In Vitro Suppression of Hepatocellular
Carcinoma by EF24, a Curcumin Analog
Haitao Liu
1.
, Yingjian Liang
1.
, Luoluo Wang
1
, Lantian Tian
1
, Ruipeng Song
1
, Tianwen Han
1
,
Shangha Pan
2
, Lianxin Liu
1
*
1Department of Hepatic Surgery, The First Affiliated Hospital of Harbin Medical University, Key Laboratory of Hepatosplenic Surgery, Ministry of Education, Harbin,
Heilongjiang Province, P.R.China, 2Research Centre of The First Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang Province, P.R.China
Abstract
The synthetic compound 3,5-bis(2-flurobenzylidene)piperidin-4-one (EF24) is a potent analog of curcumin that exhibits
enhanced biological activity and bioavailability without increasing toxicity. EF24 exerts antitumor activity by arresting the
cell cycle and inducing apoptosis, suppressing many types of cancer cells in vitro. The antiproliferative and antiangiogenic
properties of EF24 provide theoretical support for its development and application to liver cancers. We investigated the in
vitro and in vivo activities of EF24 on liver cancer to better understand its therapeutic effects and mechanisms. EF24 induced
significant apoptosis and G2/M-phase cell cycle arrest in mouse liver cancer cell lines, Hepa1-6 and H22. The expression
levels of G2/M cell cycle regulating factors, cyclin B1 and Cdc2, were significantly decreased, pp53, p53, and p21 were
significantly increased in EF24-treated cells. In addition, EF24 treatment significantly reduced Bcl-2 concomitant with an
increase in Bax, enhanced the release of cytochrome cfrom the mitochondria into the cytosol, resulting in an upregulation
of cleaved-caspase-3, which promoted poly (ADP-ribose) polymerase cleavage. EF24-treated cells also displayed decreases
in phosphorylated Akt, phosphorylated extracellular signal-regulated kinase and vascular endothelial growth factor. Our in
vitro protein expression data were confirmed in vivo using a subcutaneous hepatocellular carcinoma (HCC) tumor model.
This mouse HCC model confirmed that total body weight was unchanged following EF24 treatment, although tumor weight
was significantly decreased. Using an orthotopic HCC model, EF24 significantly reduced the liver/body weight ratio and
relative tumor areas compared to the control group. In situ detection of apoptotic cells and quantification of Ki-67, a
biomarker of cell proliferation, all indicated significant tumor suppression with EF24 treatment. These results suggest that
EF24 exhibits anti-tumor activity on liver cancer cells via mitochondria-dependent apoptosis and inducing cell cycle arrest
coupled with antiangiogenesis. The demonstrated activities of EF24 support its further evaluation as a treatment for human
liver cancers.
Citation: Liu H, Liang Y, Wang L, Tian L, Song R, et al. (2012) In Vivo and In Vitro Suppression of Hepatocellular Carcinoma by EF24, a Curcumin Analog. PLoS
ONE 7(10): e48075. doi:10.1371/journal.pone.0048075
Editor: Wael El-Rifai, Vanderbilt University Medical Center, United States of America
Received May 9, 2012; Accepted September 19, 2012; Published October 31, 2012
Copyright: ß2012 Liu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by Program for Innovative Research Team (in Science and Technology) in Higher Educational Institutions of Heilongjiang
Province (2009td06); The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: liulianxin@medmail.com.cn.
.These authors contributed equally to this work.
Introduction
Hepatocellular carcinoma (HCC) is the most common form of
primary hepatic carcinoma, the fifth most common cancer, and
the third leading cause of cancer-related deaths in the world
[1,2,3]. HCC poses a sociomedical problem particularly in Asia
and sub-Saharan Africa, where the number of deaths nearly equal
the number of cases diagnosed annually (about 600,000), and the
5-year survival rate is below 9% [4,5,6,7]. Several treatment
options exist for HCC, including resection, liver transplantation,
percutaneous ablation. However, the cure rate for patients who
undergo resection is relatively low, and among patients who are
ineligible for surgical or percutaneous procedures, only chemoem-
bolization improves survival. Moreover, HCC is widely regarded
as a chemotherapy-resistant disease [8,9,10,11,12]. These draw-
backs necessitate the continued search for novel HCC therapies.
A diverse array of phytochemicals, including some obtained
from fruits, vegetables, nuts, and spices, have demonstrated the
capacity to selectively kill tumor cells and suppress carcinogenesis
in preclinical animal models [13,14,15,16,17]. In several high-risk
populations (e.g., patients with cardiovascular disease or cancer),
phytochemicals have been shown to significantly prevent or delay
cancer development [18,19,20]. Curcumin, a polyphenol extract-
ed from rhizomes of Curcuma longa L., is a well-known
chemopreventative agent that exhibits potent anticarcinogenic
activity in a wide variety of tumor cells. Curcumin shows
significant therapeutic potential for liver cancers because it
suppresses cancer cell proliferation, induces cell cycle arrest and
apoptosis via the caspase cascade, and inhibits hypoxia-inducible
factor-1 (HIF-1) by degrading the aryl hydrocarbon receptor
nuclear translocator. Curcumin also exerts anticarcinogenic effects
by decreasing the expression of cyclooxygenase-2 (COX-2) and
vascular endothelial growth factor (VEGF) [21,22,23,24,25,26].
Unfortunately, curcumin’s therapeutic benefit is limited by very
low absorptive capacity upon transdermal or oral application [27].
Ames et al. developed a series of novel synthetic curcumin analogs
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with higher potencies and improved water solubilities [28]. One of
these compounds, EF24, exhibited approximately 10- and 20-fold
enhanced cytotoxic activity against various cancer cell lines
relative to curcumin and cisplatin, respectively [29]. EF24 induces
G2/M phase cell cycle arrest and apoptosis by increasing
phosphatase and tensin homolog (PTEN) expression in the human
ovarian carcinoma cell line, A2780. Moreover, EF24 inhibits HIF
transcriptional activity in MDA-MB231 breast cancer cells and in
PC3 prostate cancer cells. Lung cancer cell viability is decreased
by EF24 via increased phosphorylation of extracellular regulated
kinases (ERK)1/2, c-Jun N-terminal kinases (JNK), and p38
mitogen-activated protein kinases (MAPK). EF24 also inhibits the
proliferation of HCT-116 and HT-29 colon cancer cells as well as
AGS gastric adenocarcinoma cells [30,31,32,33].
Recent studies have suggested that EF24 may inhibit the
proliferation of liver cancer cells by interfering with the nuclear
factor kappa B (NF-kB) pathway. EF24 has demonstrated superior
pharmacokinetic and activity profiles in animal models and is well-
tolerated [29,34]. In addition, the compound inhibits VEGF-
induced angiogenesis in rabbit and mouse models and significantly
reduces tumor sizes in athymic nude mice xenografted with
human breast cancer tumors [35].
Considering EF24’s inhibition of ERK 1/2, upregulation of
activated caspase-3, and alteration of Bax/Bcl-2 and Bax/Bcl-xL
ratios [33,36], we examined the therapeutic potential and
molecular mechanisms of EF24 on liver cancer cells. EF24
potently induced apoptosis via mitochondrial pathway and
arrested G2/M-phase cell cycle progression in mouse liver cancer
cell lines, Hepa1-6 and H22. The expression levels of G2/M cell
cycle regulating factors, pp53, p53, and p21 were significantly
increased, cyclin B1 and Cdc2, were decreased with EF24
treatment, as were pAkt, pERK and VEGF. In vitro angiogenesis
assay and in vivo measurements of liver weight, body weight, and
tumor volume suggest that EF24 suppresses tumor growth and
induces apoptosis.
Results
EF24 Inhibits Cell Proliferation and Reduces Cell Viability
Using the CCK-8 assay, we evaluated the effect of EF24 on the
proliferation of Hepa1-6 and H22 cells. At a dose of 4 mM, EF24
exposure for 48 h strongly inhibited cell proliferation, producing
IC50 values of 4.4 mM for Hepa1-6 and 3.8 mM for H22 cells
(Fig. 1a). To evaluate whether EF24 inhibited cell viability by
inducing apoptosis, we performed an annexin V/propidium iodide
assay. Within 48 h of EF24 treatment (4 mM), 40.4% of H22 cells
and 31.8% of Hepa1-6 cells underwent apoptosis (Fig. 1b and c).
Liver cancer cells incubated with EF24 (4 mM) for 48 h were
assessed by Western blotting using antibodies that recognize the
intact (116 kDa) and cleaved (89 kDa) forms of PARP as well as
other apoptosis-related proteins. The levels of cytochrome c,
cleaved-PARP, Bax, and activated caspase-3 increased, whereas
PARP and Bcl-2 were downregulated compared with non-EF24-
treated controls (Fig. 1d and e). Treatment of Hepa1-6 and H22
cells with the pan-caspase inhibitor, z-VAD-fmk, indicated that the
apoptosis induced by EF24 was at least partly caspase-dependent
(Fig. 1f).
EF24 Induces Cell Cycle Arrest in Mouse Liver Cancer
Cells
Cell cycle analysis was performed to determine the stage at
which EF24 arrests liver cancer cells. EF24-treated (2 mM) Hepa1-
6 and H22 cells were fixed, and cell cycle distributions were
determined by flow cytometry. EF24 treatment for 48 h arrested
cells at the G2/M stage (Fig. 2a and b). Western blotting of G2/M
cell cycle regulatory molecules demonstrated that cyclin B1 and
Cdc2 were significantly reduced, pp53, p53, and p21 were
significantly increased with EF24 treatment, In addition, the level
of MDM2, one of the negative regulators of p53, was also
decreased after EF24 treatment (Fig. 2c).
EF24 Inhibits Angiogenesis and Tumor Cell Survival
Signaling in Liver Cancer
Western blot analyses of Hepa1-6 and H22 cell lysates indicated
a significant decrease in Akt and ERK phosphorylation status
following EF24 treatment (4 mM), as was COX-2, which is
involved in cell proliferation. In contrast, EF24 had no impact on
the level of total Akt, ERK (Fig. 3a), p38, or JNK (data not shown).
Human umbilical vein endothelial cells (HUVECs) were treated
with different concentrations of EF24 for 24 or 48 h, before the
cell proliferation was assessed by CCK-8 assay. As shown in
Fig. 3b, At a dose of 2 mM, EF24 exposure for 48 h inhibited cell
proliferation effectively, producing IC50 value of 3.1 mM and
2.6 mM after treatment with EF24 for 24 h and 48 h, respectively.
EF24 inhibited the proliferation of HUVECs in a dose-dependent
manner for 24 or 48 h. These results were confirmed by crystal
violet assay (Fig. 3c). These studies suggest that EF24 may exhibit
its antiangiogenic activity through inhibit the proliferation of
specific growth-related signals of vascular endothelial cell.In vivo,
EF24 significantly inhibited ERK phosphorylation (Fig. 3d). All
these findings suggesting that EF24 maybe a potent inhibitor of
tumor cell survival via PI3K/AKT, ERK-MAPK pathway
inhibition. As expected from in vitro results, VEGF and COX-2
expression levels were significantly reduced in EF24-treated
subcutaneous HCC tumor models (Fig. 3d).
EF24 Inhibits Tumor Growth and Induces Apoptosis in a
Subcutaneous HCC Tumor Model and in an Orthotopic
HCC Model
EF24 treatment significantly inhibited tumor growth in the
subcutaneous HCC model (Fig. 4a), but did not affect total body
weight, relative to controls. EF24-treated animals resulted in a
significantly lower tumor size and weight compared with controls
(Fig. 4b and c). Western blot analysis of subcutaneous tumors
excised on day 22 after EF24 treatment indicated decreased
protein expression levels of caspase-3, cyclin B1, Cdc2, and the
Bcl-2/Bax ratio, relative to controls (Fig. 4d).
In the orthotopic HCC model, mice were terminated after 3
weeks of treatment, and livers were excised (Fig. 4e). EF24-treated
animals displayed a significantly reduced liver/body weight ratio
(0.155), compared with controls (0.101) P,0.01(Fig. 4f). EF24 also
significantly reduced the relative areas of the tumors (47%)
compared with controls (72%) (Fig. 4g and h). Ki-67-based
quantification of cell proliferation in EF24-treated tumors
removed from orthotopic HCC animals indicated the presence
of more apoptotic cells and fewer Ki-67 positive cells (Fig. 4i and j).
These results suggest that EF24 significantly inhibits HCC tumor
growth and induces apoptosis in vivo.
Discussion
HCC is a common cancer typically associated with poor
prognoses, we set up to investigate novel therapies for this disease,
and find that EF24, a curcumin analogue, possesses great potential
as a promising anti-HCC therapeutic agent. EF24 induces G2/M-
phase cell cycle arrest in several different types of cancer cells,
including human breast, prostate, and cisplatin-resistant ovarian
cancer cells [30,34]. We detected G2/M-phase cell cycle arrest in
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two mouse liver cancer cells lines following treatment with EF24.
The G2/M-related proteins, cyclin B1 and Cdc2, were decreased
following treatment, while the pp53, p53, and p21, were increased.
The cyclinB1–Cdc2 complex is a key regulator of G2/M cell cycle
checkpoints. Down-regulation of cyclin B1 and Cdc2 contributes
critically to the G2/M-phase cell cycle arrest under conditions of
DNA damage. Tumor suppressor protein of p53 controls the G2/
M cell cycle checkpoints that mediate growth arrest [37], in our
studies, we found that MDM2 was decreased after treatment,
which is a negative regulator of p53. Its downregulation increases
the transactivation of p53 to promote the induction of p21, which
causes cell cycle arrest in the G2/M phase by binding of the
cyclin-cdk complex [38,39,40].
EF24 can induce cell apoptosis via the mitochondrial cell death
pathway. EF24-treated liver cancer cells expressed significantly
lower levels of Bcl-2 while concomitantly upregulating Bax
expression, compared with control cells. In addition, the release
of cytochrome cfrom the mitochondria was found to be increased
in the treatment group, which release was known to be facilitated
by Bax and blocked by Bcl-2 [41]. This dynamic upregulated
cleaved-caspase-3, promoting PARP cleavage, which is one of the
hallmarks of apoptosis [42]. Notably, EF24-induced liver cancer
cell death was not fully blocked by the general caspase inhibitor, z-
VAD-fmk, suggesting that additional mechanisms contribute to
EF24’s activity.
MAPKs including ERK, p38, and JNK, are activated by a wide
array of extracellular signals that elicit phosphorylation cascades,
transduce mitogenic signals to the nucleus, and modulate the
activity of transcription factors [43,44,45,46]. The isoforms
p42mapk (ERK-2) and p44mapk (ERK-1) are directly activated
by phosphorylation on specific tyrosine and threonine residues by
the dual-specificity ERK kinase. Because curcumin can inhibit
liver cancer cell growth by decreasing ERK activation [33,47], we
investigated whether the effect of EF24 on liver cancer cells was
associated with ERK inhibition as well. Western blot analyses of
Hepa1-6 and H22 cells lysates indicated significantly decreased
ERK phosphorylation following EF24 treatment. In contrast, p38
and JNK expression were unaffected by EF24 treatment,
suggesting that pathways other than ERK–MAPK may also be
affected by EF24 in liver cancer cells. Our next phase of research is
to examine activated ERK, which relays mitogenic signals to the
nucleus and modulates the activity of transcription factors. In
addition, we found the expression level of phospho-Akt was
decreased, without any changes in the total Akt protein level
Figure 1. EF24 inhibits cell proliferation and reduces cell viability. (a). Hepa1-6 and H22 cells were treated with the indicated concentrations
of EF24 for 48 h and 72 h. Cell growth was determined by cell counting kit-8 assay. (b). Hepa1-6 cells and H22 cells incubated with 2 mM and 4 mMof
EF24 for 48 h were analyzed for apoptosis. (c). Percentage of apoptotic cells as determined by flow cytometry of three independent experiments,
*P,0.01 compared with the untreated (DMSO) cells. (d). and (e). Lysates from Hepa1-6 and H22 cells incubated with EF24 (4 mM) were analyzed by
Western blotting for apoptosis-related proteins. (f). Hepa1-6 and H22 cells treated with EF24 and EF24 in combination with pan-caspase inhibitor z-
VAD-fmk, Cell lysates were used for immunoblot assay for cleaved caspase-3.
doi:10.1371/journal.pone.0048075.g001
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following EF24 treatment. The downregulation of pAkt resulting
in increased apoptosis and decreased cell survival as several reports
have shown that some anticancer agents induced apoptosis partly
by blocking the activation of Akt [48,49].
Tumor angiogenesis, a prerequisite for tumor growth, involves
several types of growth factors. Of these, VEGF is the most
powerful, it is a potent inducer of capillary growth into a tumor,
and without angiogenesis, tumor growth typically is limited to 1–
2 mm [33]. A study involving immunohistochemistry analysis of
24 HCC cases reported that COX-2 and VEGF expression levels
were positively correlated [50]. COX-2 inhibition induces
apoptosis signaling via death receptors in HCC [51]. In vivo,
We observed a significant decrease in both COX-2 and VEGF
expression. In vitro, we also found that treatment of HUVECs
with EF24 result in a significant inhibition of the cell proliferation,
suggesting that EF24 may exhibit the antiangiogenic activity by
inhibition of vascular endothelial cell proliferation, or this pathway
is involved in EF24-mediated HCC suppression.
To determine whether EF24 was superior to curcumin in vivo,
we designed and executed a subcutaneous HCC tumor model
using C57BL/6 mice. After 3 weeks of treatment, we measured
tumor volume and tumor weight, and observed that EF24 evoked
a significant suppression of tumor growth compared with controls,
and our novel orthotopic HCC model supports our subcutaneous
HCC tumor model. However, EF24 treatment did not affect total
body weight. Excised tumors were subsequently examined for
consistency with our in vitro protein expression results. Indeed, we
detected decreased in vivo expression of p-ERK, pro-caspase-3,
VEGF, COX-2, cyclin B1, Cdc2, and Bcl-2/Bax in the EF24-
treated group. These data support EF24 as a potential therapeutic
for liver cancer cells and indicate that EF24 functions via multiple
molecular targets to suppress cancer cell proliferation and
antiangiogenesis and induce cell cycle arrest and apoptosis.
Our results support the further development of EF24 as a liver
cancer treatment. EF24 promotes apoptosis through a cascade
reaction and induces cell cycle arrest coupled with anti-angiogen-
esis. Our study was the first to report a reduction in p-ERK
following EF24 treatment of liver cancer cells, and our orthotopic
HCC model evaluated for the first time the therapeutic effect of
EF24 on liver cancer cells. EF24 significantly inhibits liver cancer
cells in vivo and in vitro, indicating that EF24 is an anti-tumor
drug worthy of further investigation and clinical evaluation.
Materials and Methods
Animals, Cell Lines, Reagents, and Antibodies
Male C57BL/6 mice (6–8 weeks old) were purchased from the
Experimental Animal Research Center at The First Affiliated
Hospital of Harbin Medical University, China. All operative
procedures were approved by the Institutional Ethics Committee
at Harbin Medical University. All experiments were performed in
accordance with the guidelines of the Committee on the Use of
Live Animals in Teaching and Research of the Harbin Medical
University, Harbin, China. Cell lines, H22 (China Center for
Type Culture Collection, Wuhan, China), HUVEC cells (Amer-
ican Type Culture Collection, Rockville, MD, USA) were cultured
in RPMI 1640 and Hepa1-6 (from a C57BL/6 mouse hepatoma
Figure 2. EF24 induces cell cycle arrest in mouse liver cancer. Cells were treated with either DMSO (control) or EF24 (4 mM) for 48 h were
collected, flow cytometric analysis was performed for cell cycle distribution. (a). Representative flow cytometry graph for each treated or untreated
groups. (b). Distribution of G2/M phase cells. Data represent mean 6SD of three independent experiments, *p,0.05 as compared with control
group. (c). Immunoblot images of cell cycle regulatory molecules CycB1, Cdc2, p53, pp53, p21, MDM2 from treated (EF24, 4 mM, is denoted by ‘‘+’’)
and untreated (is denoted by ‘‘2’’) cells.
doi:10.1371/journal.pone.0048075.g002
Figure 3. EF24 inhibits angiogenesis and tumor cell survival signaling in liver cancer. (a). Lysates from EF24-treated (4 mM,) cells (Hepa1-6
and H22) used for western blot analysis on the expressions of ERK, p-ERK, Akt, p-Akt and COX-2. (b). HUVEC cells was treated with the indicated
concentrations of EF24 for 24 h and 48 h. Cell growth was determined by cell counting kit-8 assay. (c). The proliferation of cells was also measured by
crystal violet assay. (d). Tumor tissue lysates was used for western blotting to detect the levels of p-ERK, COX-2 and VEGF.
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[H-2b]; American Type Culture Collection, Rockville, MD, USA)
was cultured in Dulbecco’s modified Eagle’s medium (DMEM), all
supplemented with 10% fetal calf serum and 1% penicillin-
streptomycin solution at 37uC and 5% CO2. RPMI 1640
medium, DMEM, fetal calf serum, antibiotics, trypsin-EDTA,
and phosphate-buffered saline (PBS) were purchased from Gibco
Figure 4. EF24 inhibits liver cancer growth and induces apoptosis both in subcutaneous HCC tumor model and orthotopic HCC
models. (a). EF24 inhibits mice subcutaneous tumor growth in vivo. (b). Tumor size was measured after treated for three weeks. (c). EF24-treated
animals resulted in a significantly lower tumor weight when compared with controls. (d). Tumor tissue removed from the animals were used for
western blot analysis on the expressions of caspase-3, Cyclin B1, Cdc2, Bcl-2 and Bax. (e). Liver removed from the orthotopic HCC models, treated or
untreated with EF24 for three weeks. (f). EF24 treatment groups significantly reduced the liver/body weight ratio compared to the control. (g). After
treatmeant for three weeks, tumor tissue sections were stained with haematoxylin and eosion (HE). (h). Relative areas occupied by tumors in livers
were calculated. (i). Tumor sections were stained with an anti-Ki-67 Ab to detect proliferating cells. (j). Cells expressing Ki-67 were counted to
calculate the proliferation index, Assay was done in triplicate and p,0.01 is denoted by ‘‘*’’.
doi:10.1371/journal.pone.0048075.g004
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BRL (Grand Island, NY, USA). EF24 was synthesized as reported
by Adams et al. [29]. Primary antibodies against Bcl-2, Bax,
caspase-3, cleaved-caspase-3, p53, pp53, pAkt, Akt, cytochrome c,
MDM2, cyclin B1, GAPDH and secondary antibodies against
mouse IgG-horse radish peroxidase (HRP) and rabbit IgG-HRP
were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz,
CA, USA). Antibodies against PARP, p38, JNK, cleaved- poly
(ADP-ribose) polymerase (PARP), p44/42 MAPK (ERK 1/2),
phospho-p44/42 MAPK (p-T202, p-Y204) (p-ERK), Cdc2, Ki67,
and COX-2 were purchased from Cell Signaling Technology, Inc.
(Trask Lane, Danvers, MA, USA). Antibody against VEGF was
acquired from Abcam Inc. (Cambridge, MA, USA).
Cell Viability Assay
Hepal-6 and H22 cells were seeded into 96-well plates at
densities of 3610
3
cells/well and were cultured for 48 h or 72 h
in DMEM (HUVECs were cultured for 24 h or 48 h in
RPMI1640) supplemented with 10% FBS and containing EF24
at various concentrations (0, 0.5, 1.0, 2.0, 4.0, or 8.0 mM ).Cells
viability was assessed using a Cell Counting Kit-8 (CCK-8;
Dojindo Molecular Technologies, Tokyo, Japan). The cell
viability index was calculated as a percentage of the absorbance
in treated wells relative to the absorbance in untreated (control)
wells. Crystal violet assay method: HUVECs (1610
5
) were
seeded into 6-well plates and cultured overnight. Then cells was
cultured in the medium which containing EF24 (0, 2.0, or
14 mM) for 24 h or 48 h, then, cells were washed with PBS
twice, the remaining cells were stained for 1 h with crystal violet
solution (0.5% crystal violet, 20% methanol). The 6-well plates
were washed with PBS three times and left to dry at 37u,
pictures were taken. Experiments were conducted at least in
triplicate.
Apoptosis Assay
Hepal-6 and H22 cells (3610
5
cells/well) were cultured with
EF24 (2 mMor4mM) for 48 h, 1610
6
cells were collected and
washed twice with ice-cold PBS, suspended in binding buffer
(100 mL) (BD Biosciences, San Jose, CA, USA), treated with
Annexin V and Propidium iodide (PI) (BD Biosciences), and
incubated in the dark for 15 min, anther 300 mL binding buffer
were added, then flow cytometry analysis was performed within
1 h to measure the apoptosis rate (%).
Cell Cycle Analysis
To determine the effects of EF24 on the cell cycle, Hepal-6 or
H22 cells were incubated with EF24 (2 mM) for 48 h or 72 h. Cells
were trypsinized to collect both suspended and previously adhered
cells. Samples were washed twice with PBS, and the percentages of
cells in G0/G1, S, and G2/M phases were determined using a cell
cycle detection kit (BD Biosciences) in a Beckman Coulter EPICS
ALTRA II cytometer (Beckman Coulter, CA, USA).
Western Blotting
EF24-treated and -untreated Hepal-6 and H22 cells or tumor
tissues removed from subcutaneous HCC tumor model were
homogenized in protein lysate buffer, and debris was removed
by centrifugation at 12,000 rpm for 10 min at 4uC. Proteins
(50 mg/lane) were separated by 12% sodium dodecyl sulfate-
polyacrylamide gel electrophoresis. Proteins were electrotrans-
ferred onto polyvinylidene fluoride membranes (Millipore), and
membranes were washed with Tris-buffered saline (10 mM Tris,
150 mM NaCl) containing 0.05% Tween-20 and blocked with
3% bovine serum albumin. Membranes then were incubated
with primary antibody (diluted 1:1000) overnight, washed in
TBST for 30 min, exposed to HRP-conjugated secondary
antibody (diluted 1:2000), and washed again in TBST for
30 min. Final detection was performed using enhanced chemi-
luminescence (Amersham Pharmacia Biotech, Buckinghamshire,
UK).
Subcutaneous HCC Tumor Model
Approximately 2610
6
Hepa1-6 cells in 200 mL PBS were
injected subcutaneously into each animal’s back (n = 20). EF24
was dissolved in dimethyl sulfoxide and diluted in sodium chloride.
Ten days post-injection, EF24-treated mice (n = 10) were injected
intraperitoneally (i.p.) with 200 mL EF24 at a dose of 20 mg/kg/d
for 21 d. Control mice (n = 10) were injected i.p. with 200 mL PBS
daily. On day 22, tumors were removed and weighed. Tumor
volumes were estimated according to the formula p/66a
2
6b,
where a is the short axis, and b is the long axis [52].
Orthotopic HCC Model
Hepa1-6 cells were harvested following brief treatment with
0.25% trypsin and 0.2% EDTA. Trypsinization was stopped by
adding medium containing 10% FBS. Cells then were washed
in serum-free medium and resuspended in PBS. Cells with more
than 90% viability were used for the injections. Mice were
anesthetized with pentobarbital sodium, and a small left
abdominal flank incision was made. Hepa1-6 cells (2610
6
in
100 mL PBS) then were injected into the spleen parenchyma.
To avoid intrasplenic tumor growth, the spleen was removed
10 min post-injection. The incision then was closed in two
layers, using vicryl 5/0 (Warwick Medical Supplies Company
Limited, Hangzhou, China) for the abdominal wall and vicryl
4/0 for the skin. One week post-operation, the EF24-treated
group was injected i.p. with 200 ml EF24 at a dose of 20 mg/
kg/d, and control animals were injected i.p. with 200 ml PBS
daily. Mice were sacrificed at 22 d, and weights of whole bodies
and dissected livers were recorded.
Tumor-occupied Areas in Excised Livers
Excised liver tissues were embedded in paraffin, cut to 5-mm-
thick sections at 5 different liver depths, and stained with
hematoxylin and eosin. Samples were visualized under 1006
magnification (4 random fields/section; n = 20 fields). The
relative areas occupied by the tumors were calculated according
to the following formula: sum of tumor areas/20 field areas
6100% [53].
Immunohistochemistry
To quantify tumor cell proliferation, tumor tissues were
embedded in paraffin and cut to 5-mm-thick sections, immuno-
stained with anti-Ki-67 antibody as described previously [52].
Positively staining cells from 3 tumors per group were counted in
10 randomly selected fields under 4006high-power magnifica-
tion. A proliferative index (%) was calculated according to the
following formula: number of Ki-67-positive cells/total cell count
6100%.
Statistical Analysis
Data are presented as means 6standard deviations (SD) of
three independent experiments. Statistical significance was deter-
mined using Student’s t-test or Analysis of Variance (ANOVA).
Statistical significance was assigned for P,0.05.
A New Agent for Liver Cancer Treatment
PLOS ONE | www.plosone.org 7 October 2012 | Volume 7 | Issue 10 | e48075
Acknowledgments
We greatly appreciate Yuan Gao and native English-speaking experts of
BioMed Proofreading for reviewing the manuscript before submission.
Author Contributions
Conceived and designed the experiments: LL. Performed the experiments:
HL YL LW TH. Analyzed the data: LT RS. Contributed reagents/
materials/analysis tools: SP. Wrote the paper: LL.
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... Manassantin A with an IC50 value of 30 nmol/L and Manassantin B with an IC50 value of 3 nmol/L extracted from Sanbaicao are relative hypoxia-specific inhibitors of HIF-1 activation, and work by blocking hypoxia-induced accumulation of nuclear HIF-1α protein to inhibit HIF-1 activity [45]. Studies have shown that curcumin with an IC50 value of 20~50 µmol/L and its derivative EF-24 with an IC50 value of 1 µmol/L promote the degradation and inactivation of HIF-1α through the proteasome pathway, and then downregulate HIF-1 downstream target genes such as EGFR and VEGF [46,47]. ...
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Purpose Cyclooxygenase (COX)-2, an inducible enzyme which catalyzes the formation of prostaglandins from arachidonic acid, is expressed in prostate cancer specimens and cell lines. To evaluate the in vivo efficacy of a COX-2 inhibitor in prostate cancer, NS398 was administered to mice inoculated with the PC-3 human prostate cancer cell line. Materials and Methods A total of 28 male nude mice were inoculated subcutaneously with 1 million PC-3 cells. Tumors were palpable in all 28 animals 1 week after inoculation and mice were randomized to receive either vehicle (control) or NS398, 3 mg./kg. body weight, intraperitoneally three times weekly for 9 weeks. Tumors were measured at weekly intervals. After a 10-week experimental period, mice were euthanized and tumors were immuno- histochemically assayed for proliferation (PCNA), apoptosis (TUNEL) and microvessel density (MVD) (Factor-VIII-related antigen). Tumor VEGF content was assayed by Western blotting. Results NS398 induced a sustained inhibition of PC-3 tumor cell growth and a regression of existing tumors. Average tumor surface area from control mice was 285 mm.2 as compared with 22 mm.2 from treated mice (93% inhibition, p <0.001). Immunohistochemical analysis revealed that NS398 had no effect on proliferation (PCNA), but induced apoptosis (TUNEL) and decreased MVD (angiogenesis). VEGF expression was also significantly down regulated in the NS398-treated tumors. Conclusions These results demonstrate that a selective COX-2 inhibitor suppresses PC-3 cell tumor growth in vivo. Tumor growth suppression is achieved by a combination of direct induction of tumor cell apoptosis and down regulation of tumor VEGF with decreased angiogenesis
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The mitogen-activated protein kinase (MAPK) group of serine/threonine protein kinases mediates the response of cells to many extracellular stimuli such as cytokines and growth factors. These protein kinases include the extracellular signal-regulated protein kinases (ERK) and two stress-activated protein kinases (SAPK), the c-Jun N-terminal kinases (JNK), and the p38 MAPK. The enzymes are evolutionarily conserved and are activated by a common mechanism that involves a protein kinase cascade. Scaffold proteins have been proposed to interact with MAPK pathway components to create a functional signaling module and to control the specificity of signal transduction. Here we critically evaluate the evidence that supports a physiologically relevant role of MAPK scaffold proteins in mammals.
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