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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
, Yingjian Liang
, Luoluo Wang
, Lantian Tian
, Ruipeng Song
, Tianwen Han
Shangha Pan
, Lianxin Liu
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
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:
.These authors contributed equally to this work.
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.
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
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.
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.
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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.
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 ‘‘*’’.
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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
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
) 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
Apoptosis Assay
Hepal-6 and H22 cells (3610
cells/well) were cultured with
EF24 (2 mMor4mM) for 48 h, 1610
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,
Subcutaneous HCC Tumor Model
Approximately 2610
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
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
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].
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
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 | 7 October 2012 | Volume 7 | Issue 10 | e48075
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.
1. Nakashima T, Okuda K, Kojiro M, Jimi A, Yamaguchi R, et al (1983) Pathology
of hepatocellular carcinoma in Japan. 232 Consecutive cases autopsied in ten
years. Cancer 51: 863–877.
2. Jeal A, Ward E, Hao Y, Thun M (2005) Trends in the leading causes of death in
the United States, 1970–2002. Journal of the American Medical Association
294: 1255–1259.
3. Ferenci P, Fried M, Labr ecque D, Bruix J, Sherman M, et al (2010)
Hepatocellular carcinoma (hcc): a global perspective. J Clin Gastroenterol 44:
4. Bosch FX, Ribes J, Dı
´az M, Cle´ries R (2004) Primary liver cancer: worldwide
incidence and trends. Gastroenterology 127: S5–S16.
5. Sherman M (2005) Hepatocellular cancinoma: epidemiology, risk factors, and
screening. Semin Liver Dis 25: 143–154.
6. United States, National Institutes of Health, National Cancer Institute (nci).
Cancer Trends Progress Report-2009/2010 Update. Bethesda: nci; 2010.
7. Jemal A, Siegel R, Xu J, Ward E (2010) Cancer statistics. CA Cancer J Clin 60:
8. Thoppil RJ, Bishayee A (2011) Terpen oids as potential chemopreventive and
therapeutic agents in liver cancer World J Hepatol 3: 228–249.
9. Bruix J, Sherman M, Llovet JM, Beaugrand M, Lencioni R, et al (2001) Clinical
management of hepatocellular carcinoma. Conclusions of the Barcelona-2000
EASL conference. European Association for the Study of the Liver. J Hepatol
35: 421–430.
10. Lu SC (2010) Where are we in the chemoprevention of hepatocellular
carcinoma? Hepatology 51: 734–736.
11. Je Y, Schutz FA, Choueiri TK (2009) Risk of bleeding with vascular endothelial
growth factor receptor tyrosine-kinase inhibitors sunitinib and sorafenib: a
systematic review and metaanalysis of clinical trials. Lancet Oncol 10: 967–974.
12. Llovet JM, Ricci S, Mazzaferro V, Hilgard P, Gane E, et al (2008) Sorafenib in
advanced hepatocellular carcinoma. N Engl J Med 359: 378–390.
13. Aggarwal BB, Shishodia S (2006) Molecular targets of dietary agents for
prevention and therapy of cancer. Biochem Pharmacol 71: 1397–1421.
14. Russo GL (2007) Ins and outs of dietary phytochemicals in cancer
chemoprevention. Biochem Pharmacol 74: 533–544.
15. Naithani R, Huma LC, Moriarty RM, McCormick DL, Mehta RG (2008)
Comprehensive review of cancer chemopreventive agents evaluated in
experimental carcinogenesis models and clinical trials. Curr Med Chem 15:
16. Kaefer CM, Milner JA (2008) The role of herbs and spices in cancer prevention.
J Nutr Biochem 19: 347–361.
17. Moiseeva EP, Manson MM (2009) Dietary chemopreventi ve phytochemicals:
too little or too much? Cancer Prev Res (Phila) 2: 611–616.
18. Kris-Etherton PM, Hecker KD, Bonano me A, Coval SM, Binkoski AE, et al
(2002) Bioactive compounds in foods: their role in the prevention of
cardiovascular disease and cancer. Am J Med 113 Suppl 9B: 71S–88S.
19. Riboli E, Norat T (2003) Epidemiologic evidence of the protective effect of fruit
and vegetables on cancer risk. Am J Clin Nutr 78: 559S–569S.
20. World Cancer Research Fund/Ameri can Institute for Cancer Research. Food,
Nutrition, Physical Activity and the Prevention of Cancer: A Global Perspective.
Washington, DC: AICR, 2007.
21. Anand P, Sundaram C, Jhurani S, Kunnumakka ra AB, Aggarwal BB (2008)
Curcumin and cancer: an ‘‘old-age’’ disease with an ‘‘age-old’’ solution. Cancer
Lett 267: 133–164.
22. Bae MK, Kim SH, Jeong JW, Lee YM, Kim HS, et al (2006) Curcumin inhibits
hypoxia-induced angiogenesis via down-regulation of HIF-1. Oncol Rep 15:
23. Choi H, Chun YS, Kim SW, Kim MS, Park JW (2006) Curcumin inhibits
hypoxia-inducible factor-1 by degrading aryl hydrocarbon receptor nuclear
translocator: a mechanism of tumor growth inhibition. Mol Pharmacol 70:
24. Labbozzetta M, Notarbartolo M, Poma P, Giannitrapani L, Cervello M, et al
(2006) Significance of autologous interleukin-6 production in the HA22T/VGH
cell model of hepatocellular carcinoma. Ann N Y Acad Sci 1089: 268–275.
25. Aggarwal BB, Kumar A, Bharti AC (2003) Anticancer poten tial of curcumin:
preclinical and clinical studies. Anticancer Res 23: 363–398.
26. Liang Y, Yin D, Zheng T, Wang J, Meng X, et al (2011 ) Diphenyl
Difluoroketone: A Potent Chemotherapy Candidate for Human Hepatocellular
Carcinoma. Plos One 6: e23908.
27. Shoba G, Joy D, Joseph T, Majeed M, Rajendran R, et al (1998) Influence of
piperine on the pharmacokinetics of curcumin in animals and human volunteers.
Planta Med 64: 353–356.
28. Buhrow SA, Reid JM, Jia L, Mamoru SJ, Snyder JP, et al (2005) LC/MS/MS
assay and mouse pharmacokinetics and metabolism of the novel curcumin
analog EF-24 (NSC 716993). AACR Meeting Abstracts 2005 2005: 984-a.
29. Adams BK, Ferstl EM, Davis MC, Herold M, Kurtkaya S, et al (2004) Synthesis
and biological evaluation of novel curcumin analogs as anti-cancer and
antiangiogenesis agents. Bioorg Med Chem 12: 3871–3883.
30. Selvendiran K, Tong L, Vishwanath S, Bratasz A, Trigg NJ, et al (2007) EF24
induces G2/M arrest and apoptosis in cisplatin-resistant human ovarian cancer
cells by increasing PTEN expression. J Biol Chem 282: 28609–28618.
31. Thomas SL, Zhong D, Zhou W, Malik S, Liotta D, et al (2008) EF24, a novel
curcumin analog, disrupts the microtubule cytoskeleton and inhibits HIF-1. Cell
Cycle 7: 2409–2417.
32. Thomas SL, Zhao J, Li Z, Lou B, Du Y, et al (2010) Activation of the p38
pathway by a novel monoketone curcumin analog, EF24, suggests a potential
combination strategy. Biochem Pharmacol 80: 1309–1316.
33. Subramaniam D, May R, Sureban SM, Lee KB, Geor ge R, et al (2008)
Diphenyl difluoroketone: a curcumin derivative with potent in vivo anticancer
activity. Cancer Res 68: 1962–1969.
34. Adams BK, Cai J, Armstrong J, Herold M, Lu YJ, et al (2005) EF24, a novel
synthetic curcumin analog, induces apoptosis in cancer cells via a redox-
dependent mechanism. Anticancer Drugs 16: 263–275.
35. Shoji M, Sun A, Kisiel W, Lu YJ, Shim H, et al (2008) Targeting tissue factor-
expressing tumor angiogenesis and tumors with EF24 conjugated to factor VIIa.
J Drug Target 16: 185–197.
36. Zhou Y, Zheng S, Lin J, Zhang QJ, Chen A (2007) The interruption of the
PDGF and EGF signaling pathways by curcumin stimulates gene expression of
PPARcin rat activated hepatic stellate cell in vitro. Laboratory Investigation 87,
37. Mayo LD, Dixon JE, Durden DL, Tonks NK, Donner DB (2002) PTEN
Protects p53 from Mdm2 and Sensitizes Cancer Cells to Chemotherapy. J Biol
Chem. 277: 5484–5489.
38. Agarwal ML, Agarwal A, Taylor WR, Stark GR (1995) p53 controls both the
G2/M and the G1 cell cycle checkpoints and mediates reversible growth arrest
in human fibroblasts Proc. Natl Acad Sci U S A. 92: 8493–8497.
39. Deiry WS, Tokino T, Velculescu VE, Levy DB, Parsons R, et al (1993) WAF1, a
potential mediator of p53 tumor suppression. Cell 75: 817–825.
40. Coqueret O (2003) New roles for p21 and p27 cell-cycle inhibitors: a function for
each cell compartment? Trends Cell Biol 13: 65–70.
41. Kluck RM, Bossy WE, Green DR, Newmeyer DD (1997) The release of
cytochrome c from mitochondria: a primary site for Bcl-2 regulation of
apoptosis. Science 275: 1132–1136.
42. Wang X (2001) The expanding role of mitochondria in apoptosis. Genes Dev 15:
43. de Groot RP, Coffer PJ, Koenderman L (1998) Regulation of proliferation,
differentiation and survival by the IL-3/IL-5/GM-CSF receptor family. Cell
Signal 10: 619–628.
44. Malemud CJ (2007) Inhibitors of stress-activated protein/mitog en-activated
protein kinase pathways. Curr Opin Pharmacol 7: 339–343.
45. Morrison DK, Davis RJ (2003) Regulation of MAP kinase signaling modules by
scaffold proteins in mammals. Annu Rev Cell Dev Biol 19: 91–118.
46. Rennefahrt U, Janakiraman M, Ollinger R, Troppmair J (2005) Stress kinase
signaling in cancer: fact or fiction? Cancer Lett 217: 1–9.
47. Chen A, Xu J, Johnson AC (2006) Curcumin inhibits human colon cancer cell
growth by suppressing gene expression of epidermal growth factor receptor
through reducing the activity of the transcription factor, Egr-1. Oncogene 25:
48. Krystal GW, Sulanke G, Litz J (2002) Inhibition of Phosphatidylinositol 3-
Kinase-Akt Signaling Blocks Growth, Promotes Apoptosis, and Enhances
Sensitivity of Small Cell Lung Cancer Cells to Chemotherapy. Mol Cancer Ther
1: 913–922.
49. Liu X, Shi Y, Giranda VL, Luo Y (2006) Inhibition of the phosphatidylinositol
3-kinase/Akt pathway sensitizes MDA-MB468 human breast cancer cells to
cerulenin-induced apoptosis. Mol Cancer Ther 5: 494–501.
50. Liu XH, Kirschenbaum A, Yao S, Lee R, Holland JF, et al (2000) Inhibition of
cyclooxygenase-2 suppresses angiogenesis and the growth of prostate cancer in
vivo. J Urol 164: 820–825.
51. Kern MA, Hauqq AM, Koch AF, Schilling T, Breuhahn K, et al (2006)
Cyclooxygenase-2 Inhibition Induces Apoptosis Signaling via Death Receptors
and Mitochondria in Hepatocellular carcinoma. Cancer Res 66: 7059–7066.
52. Liu F, Wang P, Jiang X, Tan G, Qiao H, et al (2008) Antisense hypoxiaind ucible
factor 1alpha gene therapy enhances the therapeutic efficacy of doxorubicin to
combat hepatocellular carcinoma. Cancer Sci 99: 2055–2061.
53. Wang J, Ma Y, Jiang H, Zhu H, Liu L, et al (2011) Overexpression of von
Hippel–Lindau protein synergizes with doxorubicin to suppress hepatocellular
carcinoma in mice. Journal of Hepatology 55: 359–368.
A New Agent for Liver Cancer Treatment
PLOS ONE | 8 October 2012 | Volume 7 | Issue 10 | e48075
... 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]. ...
... Manassantin A and Manassantin B have strong selective inhibitory effects on human breast cancer T47D cells, and also have strong inhibitory effects on secretion of hypoxia-induced VEGF, CDKN1A, and GLUT-1 genes [45,74]. Studies have shown that curcumin and its derivative EF-24 have the effect of inhibiting tumor blood-vessel growth and delaying tumor growth [46,47]. YC-1 is a targeted HIF-1α inhibitor, which can resist intravascular thrombosis, block the hypoxia signaling pathway of cells, inhibit the expressions of HIF-1α and VEGF, inhibit tumor angiogenesis, and tumor cell proliferation, thereby exerting anti-tumor and antiangiogenic effects [75,76]. ...
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Hypoxia-inducible factor-1α (HIF-1α) is widely distributed in human cells, and it can form different signaling pathways with various upstream and downstream proteins, mediate hypoxia signals, regulate cells to produce a series of compensatory responses to hypoxia, and play an important role in the physiological and pathological processes of the body, so it is a focus of biomedical research. In recent years, various types of HIF-1α inhibitors have been designed and synthesized and are expected to become a new class of drugs for the treatment of diseases such as tumors, leukemia, diabetes, and ischemic diseases. This article mainly reviews the structure and functional regulation of HIF-1α, the modes of action of HIF-1α inhibitors, and the application of HIF-1α inhibitors during the treatment of diseases.
... To solve this problem, many new analogs were synthesized, among which EF24 is an excellent agent. EF24 shows enhanced bioavailability and more potent bioactivity, such as inhibiting the proliferation, movement and epithelial-mesenchymal transition of cancer cells [9,10]. It also can induce cancer cell apoptosis and inhibit the metastasis of human tumor xenografts [10,11]. ...
... EF24 shows enhanced bioavailability and more potent bioactivity, such as inhibiting the proliferation, movement and epithelial-mesenchymal transition of cancer cells [9,10]. It also can induce cancer cell apoptosis and inhibit the metastasis of human tumor xenografts [10,11]. However, the role of EF24 in NSCLC remains unclear. ...
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Background The role of Diphenyldifluoroketone (EF24), a synthetic analogue of curcumin with noteworthy antitumor potential, remains unclear in non-small cell lung cancer (NSCLC). Herein, the inhibitory effect of EF24 on NSCLC and its mechanism were studied. Methods Cytotoxicity was measured by MTT assay, colony formation assay and xenograft model. Cell apoptosis and reactive oxygen species (ROS) level were quantified by flow cytometer. Protein level was detected by western blot assay. Mitochondria and autophagosomes were observed using transmission electron microscope and confocal microscopy. Results In-vitro, EF24 significantly induced proliferation inhibition, apoptosis, mitochondrial fission and autophagy of NSCLC cell lines. These cytotoxic effects were significantly attenuated by two reactive oxygen species (ROS) scavengers, indicating its anti-cancer effects largely depend on ROS accumulation. In-vivo, EF24 inhibited tumor growth in a dose-dependent manner. Moreover, no pathological changes of heart, lung, spleen, kidney and liver of mice were observed. Collectively, EF24 induced ROS accumulation, in turn activates cell apoptosis, and then exerts its cytotoxicity on NSCLC cells. Conclusions The results showed that EF24 exerted cytotoxicity against NSCLC via ROS accumulation. Thus, EF24 might serve as a potential anti-cancer agent for the treatment of NSCLC.
... EF-24 is a synthetic fluorinated analogue of curcumin with improved antiproliferative properties against cancer cells both in vitro and in animal models in vivo [86][87][88][89][90]. Anticancer effects of EF-24 are attributed to the suppression of NF-κB activity and by deregulation of oncogenic signalling pathways that include PTEN, Akt, and HIF-1α in cancer cell lines in vitro [87,[90][91][92]. ...
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In laboratory experiments, many electrophilic cytotoxic agents induce cell death accompanied by reactive oxygen species (ROS) production and/or by glutathione (GSH) depletion. Not surprisingly, millimolar concentrations of N-acetylcysteine (NAC), which is used as a universal ROS scavenger and precursor of GSH biosynthesis, inhibit ROS production, restore GSH levels, and prevent cell death. The protective effect of NAC is generally used as corroborative evidence that cell death induced by a studied cytotoxic agent is mediated by an oxidative stress-related mechanism. However, any simple interpretation of the results of the protective effects of NAC may be misleading because it is unable to interact with superoxide (O2•−), the most important biologically relevant ROS, and is a very weak scavenger of H2O2. In addition, NAC is used in concentrations that are unnecessarily high to stimulate GSH synthesis. Unfortunately, the possibility that NAC as a nucleophile can directly interact with cytotoxic electrophiles to form non-cytotoxic NAC–electrophile adduct is rarely considered, although it is a well-known protective mechanism that is much more common than expected. Overall, apropos the possible mechanism of the cytoprotective effect of NAC in vitro, it is appropriate to investigate whether there is a direct interaction between NAC and the cytotoxic electrophile to form a non-cytotoxic NAC–electrophilic adduct(s).
... The relative insensitivity of peripheral blood mononuclear cells and immortalized epithelial cells compared with cancer cell lines suggested selectivity for cancer and a workable therapeutic window (5). Multiple studies have since shown activity in various models for hematologic and solid tumors notably including Ewing's sarcoma, bortezomibresistant multiple myeloma, and mantle cell lymphoma which are associated with poor prognosis (5)(6)(7)(8)(9)(10)(11)(12)(13)(14). ...
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Dienone compounds have been demonstrated to display tumor-selective anti-cancer activity independently of the mutational status of TP53. Previous studies have shown that cell death elicited by this class of compounds is associated with inhibition of the ubiquitin-proteasome system (UPS). Here we extend previous findings by showing that the dienone compound b-AP15 inhibits proteasomal degradation of long-lived proteins. We show that exposure to b-AP15 results in increased association of the chaperones VCP/p97/Cdc48 and BAG6 with proteasomes. Comparisons between the gene expression profile generated by b-AP15 to those elicited by siRNA showed that knock-down of the proteasome-associated deubiquitinase (DUB) USP14 is the closest related to drug response. USP14 is a validated target for b-AP15 and we show that b-AP15 binds covalently to two cysteines, Cys203 and Cys257, in the ubiquitin-binding pocket of the enzyme. Consistent with this, deletion of USP14 resulted in decreased sensitivity to b-AP15. Targeting of USP14 was, however, found to not fully account for the observed proteasome inhibition. In search for additional targets, we utilized genome-wide CRISPR/Cas9 library screening and Proteome Integral Solubility Alteration (PISA) to identify mechanistically essential genes and b-AP15 interacting proteins respectively. Deletion of genes encoding mitochondrial proteins decreased the sensitivity to b-AP15, suggesting that mitochondrial dysfunction is coupled to cell death induced by b-AP15. Enzymes known to be involved in Phase II detoxification such as aldo-ketoreductases and glutathione-S-transferases were identified as b-AP15-targets using PISA. The finding that different exploratory approaches yielded different results may be explained in terms of a “target” not necessarily connected to the “mechanism of action” thus highlighting the importance of a holistic approach in the identification of drug targets. We conclude that b-AP15, and likely also other dienone compounds of the same class, affect protein degradation and proteasome function at more than one level.
... b Schematic diagram showing the action of Janus magnetic mesoporous silica nanocarriers for magnetically targeted and hyperthermia-enhanced curcumin therapy of liver cancer. Adapted with permission from ref. [134], copyright@2018 (RSC). c (i and ii) Effects of curcumin on the hepatic GRP78 protein expression and the ratio of p-PERK/PERK and-IRE1α/IRE1α in alcohol-induced liver injury. ...
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Purpose Among all forms of cancers, hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide. There are several treatment options for HCC ranging from loco-regional therapy to surgical treatment. Yet, there is high morbidity and mortality. Recent research focus has shifted towards more effective and less toxic cancer treatment options. Curcumin, the active ingredient in the Curcuma longa plant, has gained widespread attention in recent years because of its multifunctional properties as an antioxidant, anti-inflammatory, antimicrobial, and anticancer agent. Methods A systematic search of PubMed, Embase and Google Scholar was performed for studies reporting incidence of HCC, risk factors associated with cirrhosis and experimental use of curcumin as an anti-cancer agent. Results This review exclusively encompasses the anti-cancer properties of curcumin in HCC globally and it’s postulated molecular targets of curcumin when used against liver cancers. Conclusions This review is concluded by presenting the current challenges and future perspectives of novel plant extracts derived from C. longa and the treatment options against cancers.
... For the last >20 years, the laboratory of Jonathan Dimmock has described a large number of 1,5-diaryl-3-oxo-1,4-pentadienyl compounds and showed them to be strong inducers of tumor cell apoptosis, demonstrating a preferential activity on tumor cells over normal cells [3]. The compound EF24 (NSC716993, Figure 1) was shown to induce cell cycle arrest, apoptosis of cancer cells [21], and antineoplastic effects in animal models [22,23]. The properties of some compounds in this series are displayed in Table 1. ...
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Dienone compounds with a 1,5-diaryl-3-oxo-1,4-pentadienyl pharmacophore have been widely reported to show tumor cell selectivity. These compounds target the ubiquitin-proteasome system (UPS), known to be essential for the viability of tumor cells. The induction of oxidative stress, depletion of glutathione, and induction of high-molecular-weight (HMW) complexes have also been reported. We here examined the response of acute myeloid leukemia (AML) cells to the dienone compound VLX1570. AML cells have relatively high protein turnover rates and have also been reported to be sensitive to depletion of reduced glutathione. We found AML cells of diverse cytogenetic backgrounds to be sensitive to VLX1570, with drug exposure resulting in an accumulation of ubiquitin complexes, induction of ER stress, and the loss of cell viability in a dose-dependent manner. Caspase activation was observed but was not required for the loss of cell viability. Glutathione depletion was also observed but did not correlate to VLX1570 sensitivity. Formation of HMW complexes occurred at higher concentrations of VLX1570 than those required for the loss of cell viability and was not enhanced by glutathione depletion. To study the effect of VLX1570 we developed a zebrafish PDX model of AML and confirmed antigrowth activity in vivo. Our results show that VLX1570 induces UPS inhibition in AML cells and encourage further work in developing compounds useful for cancer therapeutics.
... Natural compounds-based photosensitizers have been studied due to their low potential to trigger side effects and drug interactions [33][34][35][36]. Curcumin is a natural compound obtained from Curcuma longa, which has a yellow coloration and widely used as a culinary spice and flavoring agent [14,[37][38][39][40][41]. It is reported that curcumin has antineoplastic, antimicrobial, anti-inflammatory and antioxidant properties and those effects are potentiated when associated to visible light [37,38,42,43]. ...
Sono-photodynamic therapy (SPDT) is a combined therapy which employs the use of a light source and ultrasound activation for antimicrobial purposes. This study evaluated the antibacterial effect of curcumin-mediated SPDT against Streptococcus mutans biofilm. Absorption spectra of curcumin was determined under UV-visible region (200-800 nm). The generation of reactive oxygen species was evaluated using a fluorescence probe (singlet oxygen sensor green). Minimum inhibitory and minimum bacterial concentrations were determined. S. mutans biofilm was cultured and treated according to the groups as follows: L - PS - U- (negative control), chlorhexidine (positive control), L + PS + U- (aPDT groups), L - PS + U+ (SDT groups), L + PS + U+ (SPDT groups). Before irradiation, the biofilms were incubated for 5 min and irradiated by blue light emitting-diode at 15 J cm-2. For the dark toxicity, the groups were exposed to the same conditions, but no light was used. After treatments, counting of colonies forming units was performed. Confocal microscopy images were obtained. Data were statistically analyzed by ANOVA and Tukey's test (p 0.05). Curcumin under 80 µM showed higher absorption than 40 µM. For the generation of reactive species, antimicrobial photodynamic therapy and SPDT exhibited similar behavior. Minimum inhibitory concentration and minimum bactericidal concentration were 40 and 80 µM, respectively. Curcumin under 40 and 80 µM showed no dark toxicity. SPDT showed better results than isolated therapies, since significant reduction (3.8 log10; p 0.001) over the cell viability was observed. This result was confirmed by confocal images. Curcumin showed promising results as a photosensitizer for SPDT. Moreover, curcumin-mediated SPDT exhibited enhanced antibacterial effects and may be an alternative therapy to control the oral biofilm.
Hepatic cancer is one of the most deadly malignancies owing to the pivotal role of liver in physiological homeostasis. Various strategies are being implemented to combat the onset and progression of liver cancer that has achieved promising success. However, conventional therapeutic approaches have their own limitations, particularly nonspecific toxicity and the onset of the chemoresistance. Therefore alternative approaches, including bioactive components of natural origins are being explored for their antineoplastic activity. Curcumin, the yellow pigment of turmeric spice, has shown effective cytotoxic activity against numerous malignant cells, including hepatic cancer. The abilities of curcumin such as its antioxidant nature, antiinflammatory effects, immunostimulatory activity, and protective behavior against organ damage make this phytochemical as a better choice of therapeutic agent in various medical ailments including malignancies of hepatic origin. Mechanistic explorations on curcumin have identified various molecular targets for its therapeutic effects against liver cancer. Moreover, curcumin is devoid of any specific adverse effects and safe for consumption. Curcumin also exhibits chemosensitizing ability and makes liver cancer cells more susceptible to conventional chemotherapeutic drugs. Although few concerns, including bioavailability and its metabolism limit the optimal clinical exploitation of curcumin, its derivatives is found to overcome such obstacles. Collectively curcumin stands high in prospective therapeutic molecules against liver cancers with evident success in preclinical as well clinical investigations.
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Diarylpentanoids exhibit a high degree of anti-cancer activity and stability in vitro over curcumin in prostate cancer cells. Hence, this study aims to investigate the effects of a diarylpentanoid, 1,5-bis(4-hydroxy-3-methoxyphenyl)-1,4-pentadiene-3-one (MS13) on cytotoxicity, anti-proliferative, apoptosis-inducing, anti-migration properties, and the underlying molecular mechanisms on treated androgen-independent prostate cancer cells, DU 145 and PC-3. A cell viability assay has shown greater cytotoxicity effects of MS13-treated DU 145 cells (EC50 7.57 ± 0.2 µM) and PC-3 cells (EC50 7.80 ± 0.7 µM) compared to curcumin (EC50: DU 145; 34.25 ± 2.7 µM and PC-3; 27.77 ± 6.4 µM). In addition, MS13 exhibited significant anti-proliferative activity against AIPC cells compared to curcumin in a dose- and time-dependent manner. Morphological observation, increased caspase-3 activity, and reduced Bcl-2 protein levels in these cells indicated that MS13 induces apoptosis in a time- and dose-dependent. Moreover, MS13 effectively inhibited the migration of DU 145 and PC-3 cells. Our results suggest that cell cycle-apoptosis and PI3K pathways were the topmost significant pathways impacted by MS13 activity. Our findings suggest that MS13 may demonstrate the anti-cancer activity by modulating DEGs associated with the cell cycle-apoptosis and PI3K pathways, thus inhibiting cell proliferation and cell migration as well as inducing apoptosis in AIPC cells.
ead the full text About Share on Abstract Gastrointestinal (GI) cancers with a high global prevalence are a leading cause of morbidity and mortality. Accordingly, there is a great need to develop efficient therapeutic approaches. Curcumin, a naturally occurring agent, is a promising compound with documented safety and anticancer activities. Recent studies have demonstrated the activity of curcumin in the prevention and treatment of different cancers. According to systematic studies on curcumin use in various diseases, it can be particularly effective in GI cancers because of its high bioavailability in the gastrointestinal tract. Nevertheless, the clinical applications of curcumin are largely limited because of its low solubility and low chemical stability in water. These limitations may be addressed by the use of relevant analogues or novel delivery systems. Herein, we summarize the pharmacological effects of curcumin against GI cancers. Moreover, we highlight the application of curcumin's analogues and novel delivery systems in the treatment of GI cancers.
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Despite significant advances in medicine, liver cancer, predominantly hepatocellular carcinoma remains a major cause of death in the United States as well as the rest of the world. As limited treatment options are currently available to patients with liver cancer, novel preventive control and effective therapeutic approaches are considered to be reasonable and decisive measures to combat this disease. Several naturally occurring dietary and non-dietary phytochemicals have shown enormous potential in the prevention and treatment of several cancers, especially those of the gastrointestinal tract. Terpenoids, the largest group of phytochemicals, traditionally used for medicinal purposes in India and China, are currently being explored as anticancer agents in clinical trials. Terpenoids (also called "isoprenoids") are secondary metabolites occurring in most organisms, particularly plants. More than 40 000 individual terpenoids are known to exist in nature with new compounds being discovered every year. A large number of terpenoids exhibit cytotoxicity against a variety of tumor cells and cancer preventive as well as anticancer efficacy in preclinical animal models. This review critically examines the potential role of naturally occurring terpenoids, from diverse origins, in the chemoprevention and treatment of liver tumors. Both in vitro and in vivo effects of these agents and related cellular and molecular mechanisms are highlighted. Potential challenges and future directions involved in the advancement of these promising natural compounds in the chemoprevention and therapy of human liver cancer are also discussed.
No effective systemic therapy exists for patients with advanced hepatocellular carcinoma. A preliminary study suggested that sorafenib, an oral multikinase inhibitor of the vascular endothelial growth factor receptor, the platelet-derived growth factor receptor, and Raf may be effective in hepatocellular carcinoma. Methods In this multicenter, phase 3, double-blind, placebo-controlled trial, we randomly assigned 602 patients with advanced hepatocellular carcinoma who had not received previous systemic treatment to receive either sorafenib (at a dose of 400 mg twice daily) or placebo. Primary outcomes were overall survival and the time to symptomatic progression. Secondary outcomes included the time to radiologic progression and safety. Results At the second planned interim analysis, 321 deaths had occurred, and the study was stopped. Median overall survival was 10.7 months in the sorafenib group and 7.9 months in the placebo group (hazard ratio in the sorafenib group, 0.69; 95% confidence interval, 0.55 to 0.87; P
In a cell-free apoptosis system, mitochondria spontaneously released cytochrome c, which activated DEVD-specific caspases, leading to fodrin cleavage and apoptotic nuclear morphology. Bcl-2 acted in situ on mitochondria to prevent the release of cytochrome c and thus caspase activation. During apoptosis in intact cells, cytochrome c translocation was similarly blocked by Bcl-2 but not by a caspase inhibitor, zVAD-fmk. In vitro, exogenous cytochrome c bypassed the inhibitory effect of Bcl-2. Cytochrome c release was unaccompanied by changes in mitochondrial membrane potential. Thus, Bcl-2 acts to inhibit cytochrome c translocation, thereby blocking caspase activation and the apoptotic process.
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
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.
The pathologic findings of 232 consecutive cases of hepatocellular carcinoma (HCC) autopsied during the past ten years at Kurume, Japan, were analyzed from the point of view of global epidemiology, in relation to clinical feature, and in regard to incidence, age, sex, etiologic factors, size of liver, changes in noncancer parenchyma, gross type of tumor, extrahepatic metastases, intravascular and intraductal growths, cancer cell histology, hepatitis B surface antigen (HBsAg) in hepatocytes and cancer cells, liver cell dysplasia, and frequency and clinicopathologic characteristics of minute HCC. Furthermore, postmortem hepatic arteriography and portography were done in 152 livers for comparison with gross anatomy and celiac angiograms. It was found that: (1) epidemiologicall), HCC in Japan is distinct from that in the West that it is frequently encapsulated, livers are generally small because of frequent and advanced cirrhosis and small cancer, minute HCC, is not uncommon at autopsy, cirrhosis most commonly associated is the one with thin stroma and medium size nodules, and micronodular cirrhosis is very rare despite frequent alcohol abuse; (2) HCC is increasing in incidence; (3) HBsAg is frequently found in parenchyma; (4) liver cell dysplasia is indirectly related to HBsAg with no evidence for premalignancy; (5) the lung is the most frequent site of metastasis but peritoneal dissemination is unusual; (6) intraportal tumor growth is very common and the hepatic vein is less frequently affected; (7) growth in the major bile duct is frequently associated with intraportal growth and clinically presents as obstructive jaundice; and (8) tumor is supplied solely by arteries and celiac arteriograms are closely correlated with gross pathologic findings.