Histone Deacetylase Inhibitors Facilitate Dihydroartemisinin-Induced Apoptosis in Liver Cancer In Vitro and In Vivo
et al. (2012) Histone Deacetylase Inhibitors Facilitate Dihydroartemisinin-Induced Apoptosis in Liver Cancer In
Vitro and In Vivo. PLoS ONE 7(6): e39870. doi:10.1371/journal.pone.0039870
Histone Deacetylase Inhibitors Facilitate Dihydroartemisinin-Induced Apoptosis in Liver Cancer In Vitro and In Vivo
Chris Zhiyi Zhang 0
Yinghua Pan 0
Yun Cao 0
Paul B. S. Lai 0
Lili Liu 0
George Gong Chen 0
Jingping Yun 0
Wael El-Rifai, Vanderbilt University Medical Center, United States of America
0 1 State Key Laboratory of Oncology in Southern China, Sun Yat-Sen University Cancer Center , Guangzhou , China , 2 Department of Pathology, Sun Yat-Sen University Cancer Center , Guangzhou , China , 3 Department of Rheumatology and Immunology, The Third Affiliated Hospital of Sun Yat-Sen University , Guangzhou , China , 4 Department of Surgery, Prince of Wales Hospital, The Chinese University of Hong Kong , Shatin, N.T. , Hong Kong
Liver cancer ranks in prevalence and mortality among top five cancers worldwide. Accumulating interests have been focused in developing new strategies for liver cancer treatment. We have previously showed that dihydroartemisinin (DHA) exhibited antitumor activity towards liver cancer. In this study, we demonstrated that histone deacetylase inhibitors (HDACi) significantly augmented the antineoplastic effect of DHA via increasing apoptosis in vitro and in vivo. Inhibition of ERK phosphorylation contributed to DHA-induced apoptosis, due to the fact that inhibitor of ERK phosphorylation (PD98059) increased DHA-induced apoptosis. Compared with DHA alone, the combined treatment with DHA and HDACi reduced mitochondria membrane potential, released cytochrome c into cytoplasm, increased p53 and Bak, decreased Mcl-1 and pERK, activated caspase 3 and PARP, and induced apoptotic cells. Furthermore, we showed that HDACi pretreatment facilitated DHA-induced apoptosis. In Hep G2-xenograft carrying nude mice, the intraperitoneal injection of DHA and SAHA resulted in significant inhibition of xenograft tumors. Results of TUNEL and H&E staining showed more apoptosis induced by combined treatment. Immunohistochemistry data revealed the activation of PARP, and the decrease of Ki-67, p-ERK and Mcl-1. Taken together, our data suggest that the combination of HDACi and DHA offers an antitumor effect on liver cancer, and this combination treatment should be considered as a promising strategy for chemotherapy.
Funding: This work was supported by grants from the National Natural Science Foundation of China (No. 81172345 and No. 30973506). 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.
Liver cancer is the fifth most common cancer worldwide and
the third most common cause of death from cancer . More than
75% of new cases are diagnosed in developing countries; however,
incidence is increasing in economically developed regions,
including Japan, Western Europe, and the United States [2,3].
Although surgical resection and liver transplant are the two major
therapeutic options with curative potential, surgery is only feasible
for about 20% of liver cancer cases since patients are most often
diagnosed at an advanced stage [4,5]. To date, chemotherapy for
liver cancer is not satisfactory and the long-term survival of liver
cancer patients is still poor [4,6]. Therefore, developing novel and
effective therapeutic strategies for liver cancer is of great need and
Histone deacetylase inhibitors (HDACi) are currently a major
focus of interest as antineoplastic agents [7,8]. HDACi is a class of
agents that function via blocking histone deacetylation, thereby
modifying chromatin structure and gene transcription .
Particularly, HDACi inhibit the acetylation of lysine residues at
the histone N-terminal tail which results in loosening the
association of histones with DNA, thereby allowing the expression
of genes related to tumor suppression . Understanding the
association between HDAC activities and various cancers led
many researchers to consider HDAC inhibitors as potent agents
that can interfere with cancer cell proliferation and/or survival
through the modulation of cell cycle progression, differentiation,
or by promoting cell death. For example, Kim et al. reported that
CG0006 exposure in breast cancer cell resulted in cell death via
down-regulation HDAC6 . Bommi et al. demonstrated that
sodium butyrate induced apoptosis in cancer cells by
transcriptional downregulation of BMI1 .
Although HDACi alone may be clinically useful, they will most
likely be of value in combination with other antitumor agents.
SAHA has been approved by the U.S. Food and Drug
Administration (FDA) for the treatment of cutaneous T cell
lymphoma and other HDACi are now undergoing Phase I/II
clinical trials as a single agent or in combination with other agents
[13,14]. Accumulating reports have been indicated the synergistic
effect on lethality of combination of HDACi and other
chemotherapeutic agents. Kretzner et al. showed that combination of
HDACi and Aki enhanced lymphoma cell death through
repression of c-Myc, hTERT, and microRNA levels . Nguyen
et al. reported that coadministration of HDACi synergistically
increased KW-2449 lethality resulting from inactivation of Bcr/
Abl . Lately, a phase II study revealed that treatment of
vorinostat combined with tamoxifen significantly prolonged the
survival of patients with breast cancer . However, such
a synergistic effect has rarely been demonstrated in liver cancer.
Recently, we have reported that Dihydroartemisinin (DHA), the
main active metabolite of artemisinin derivatives, exhibited
anticancer activity towards liver cancer . In the present study,
we showed that (a) DHA induced apoptosis via downregulating
ERK phosphorylation, which was further confirmed by the data
that the inhibitor of ERK phosphorylation (PD98059) increased
DHA-induced apoptosis, (b) HDACi in vitro remarkably enhanced
DHA-induced cell death, accompanying with reduction of
mitochondria membrane potential, release of cytochrome c into
cytoplasm, increase of p53 and Bak, and decreases of Mcl-1 and
pERK, (c) the combination of HDACi and DHA in vivo significantly
halted the growth of liver cancer tumor xenograft. Our data may
suggest the combination of HDACi and DHA as a promising
strategy for liver cancer chemotherapy.
Materials and Methods
Human liver cancer cell lines (Hep G2 and PLC/PRF/5) were
purchased from American Type Culture Collection (ATCC,
Manassas, VA) and cultured in Dulbeccos modified Eagles
medium (DMEM) (Gibco, Gaithersburg, MD) containing 10%
fetal bovine serum (FBS), 100 mg/ml penicillin, and 100 mg/ml
streptomycin in a humidified atmosphere of 5% CO2 and 95% air
Antibodies and reagents
Antibodies for Mcl-1, PARP, Bak, and Actin were purchased
from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies for
caspase 3, p38, p-p38, ERK, p-ERK, JNK, and p-JNK were
provided by Cell Signaling (Danvers, MA). Dihydroartemisinin
(DHA, dissolved in DMSO), sodium butyrate (NaB, dissolved in
H2O), suberoylanilide hydroxamic acid (SAHA, dissolved in
DMSO) and p-ERK inhibitor (PD98059, dissolved in DMSO)
were purchased from Sigma (St. Louis, MO).
Cell viability was assessed by 3-(4, 5-dimethylthiazol-2-yl)-2,
5diphenyltetrazo-lium bromide (MTT) assay. Briefly, 86103 of cells
were seeded into 96-well plates for 24 h, followed by incubation
with various doses of DHA for indicated time. After adding
100 ml/well of MTT solution, the cells were incubated for another
2 h. Supernatants were then removed and the formazan crystals
were dissolved in 100 ml/well DMSO. The absorbance at 570/
630 nm of each sample was measured using multilabel plate
reader (PerkinElmer). Three independent experiments were
One hundred of cells were seeded into 6-well plates, and
cultured for 7 d. And then the medium was replaced by fresh one
containing DHA. After being incubated for another 7 d, colony
formed by liver cancer cells was stained with 0.05% crystal violet
(Sigma, St. Louis, MO) for 8 min. The number of colony was then
Cell lysates were boiled with 6x sodium dodecyl sulfate (SDS)
loading buffer and then fractionated by SDS-PAGE. The proteins
were transferred to PVDF membrane which was then incubated
with a primary specific antibody in 5% of non-fat milk, followed by
a horse radish peroxidase (HRP)-conjugated anti-mouse or
antirabbit second antibodies. ECL detection reagent (Amersham Life
Science, Piscataway, NJ) was used to demonstrate the results.
Annexin V/PI assay
Apoptosis was assessed using Annexin V-PI double staining.
After treatments, cells were trypsinized, and stained with 0.5 mg/
ml Annexin V in binding buffer (10 mM HEPES free acid,
0.14 M NaCl, and 2.5 mM CaCl2) for 30 min. Afterward, PI
(5 mg/mL final concentration) was added and incubated for
another 15 min. Cells were applied to a flow cytometer for data
Apoptosis assay was performed using Apo-Direct TUNEL
Assay kit (Millipore). Cells were harvested and fixed in 4% PFA for
60 min at 4uC, followed by a second fixation in 70% (v/v) ethanol
overnight at 220uC. Cells were then treated with various reagents
for a designed period according to the manufactures instruction.
Finally, cells were analyzed by flow cytometry using FACS
Vantage machine (Becton Dickinson). The Cell Quest software
(Verity Software House) was used to analyze the data.
In situ cell death detection
Labeling of fragmented DNA to assess apoptosis was performed
with TUNEL staining (green fluorescence), using In Situ Cell
Death Detection Kit (Roche, LA), as described in our previous
Measurement of mitochondrial membrane potential
(Dym) by flow cytometry
Forty nM of DioC6 (SigmaAldrich, MO) were incubated with
treated cells at indicated time points for 15 min at 37uC. The
harvested cells were washed with ice-cold PBS and analyzed by
flow cytometry using Becton Dickinson FACS Vantage machine
(Becton Dickinson, NJ). Cells with low Dym were presented as
a percentage of the total cell population. The CellQuest software
(Verity Software House) was used to analyze the data.
All animal experiments were conducted according to relevant
national and international guidelines and have been approved by
the Institute Research Medical Ethics Committee of Sun Yat-Sen
University Cancer Center. 16107 of Hep G2 cells were suspended
in sterile PBS and injected subcutaneously into the right flank of
the mice. Mice were checked daily for xenograft/tumor
development. Mice were randomized into three groups of 6 mice/
group. DHA (5 mg/kg mouse body weight) was given to the
DHA group, SAHA (1.5 mg/kg mouse body weight) was given to
the SAHA group, combination of SAHA and DHA was given to
DHA+SAHA group, once daily for five consecutive days per
week for 24 d. The DMSO group received an equal volume of
solvent control. After treatment at various time intervals, mouse
body weight and tumor size were measured. Finally, tumors were
excised, weighed and fixed in 4% of PFA. Paraffin-embedded
Formalin-fixed and paraffin-embedded liver cancer sections
with a thickness of 4 mm were dewaxed in xylene and graded
alcohols, hydrated, and washed in phosphatebuffered saline (PBS).
After pretreatment in a microwave oven, endogenous peroxidase
was inhibited by 3% hydrogen peroxide in methanol for 20 min,
followed by avidin-biotin blocking using a biotin-blocking kit
(DAKO, Germany). Slides were then incubated with antibodies
for 4 h in a moist chamber at room temperature, washed in PBS,
and incubated with biotinylated goat anti-rabbit/mouse
antibodies. Slides were developed with the Dako Liquid 3,
3diaminobenzidine tetrahydrochloride (DAB) +Substrate
Chromogen System and counterstained with hematoxylin.
Difference between groups was determined for statistical
significance using one-way ANOVA or Students t-test. All
Pvalues are two-sided and P,0.05 was considered as statistically
significant. All statistical calculations were performed with the
SPSS software (SPSS, Inc., Chicago, IL). The data were presented
as mean6SD from at least three independent experiments.
Activations of MAP kinases were involved in
DHA has been demonstrated to induce cell death in human
cancers [20,21]. We first assessed DHA-induced apoptosis in liver
cancer cell lines, using Annexin V assay. Results indicated that
percentage of Annexin V-positive cells were dramatically
increased upon DHA treatment (Fig. 1A), suggesting DHA being
a potent apoptosis inducer in liver cancer cells. Compared to the
control, following exposure of 10 mM DHA for 24 h, the
percentage of apoptotic cells was remarkably increased from
5.3% and 4.9% to 16.6% and 13.5%, respectively in Hep G2 and
PLC/PRF/5 cells (Fig. 1B).
Apoptosis induction usually associates with activation of MAP
kinases. A time-course analysis was performed on the
phosphorylation levels of three MAP kinase members, including
extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase
(JNK) and p38 (Fig. 1C). The protein levels of all 3 MAP kinases
remained unchanged. However, p38 phosphorylation was
increased after DHA treatment in both tested cells. The level of JNK
phosphorylation remained the same as control in Hep G2 cells,
but was markedly increased in PLC/PRF/5 cells (Fig. 1D). The
levels of p-ERK appeared decreasing in both liver cancer cells
treated with DHA. These data may suggest that inactivation of
ERK contributes to DHA-induced apoptosis.
Inhibition of ERK phosphorylation was attributed to
DHA-induced apoptosis in liver cancer cells
To test our assumption that inactivation of ERK was involved
in DHA-induced apoptosis, we pretreated cells with PD98059, an
inhibitor of ERK phosphorylation. Firstly, the cytotoxicity of
PD98059 was tested. Results indicated that PD98059 alone did
not cause significant apoptosis in both cells (data not shown). We
next determined the effect of PD98059 on DHA-induced cell
growth attenuation. As indicated by MTT result, PD98059
significantly reduced liver cancer cell viabilities, compared with
DHA groups (Fig. 2A).
Next, we examined whether PD98059 treatment enhanced
DHA-induced cell growth inhibition through inducing apoptosis.
We assessed DHA-induced apoptosis in liver cancer cells
pretreated with 10 mM PD98059 for 2 h by Hoechst 33342
staining. Results revealed more cells with characteristic features
including chromatin condensation and apoptotic body presented
in PD98059-pretreated cells (Fig. 2B). This was further confirmed
by TUNEL assays showing that the percentages of
TUNELpositive cells were increased in liver cancer cells treated with both
PD98059 and DHA (Fig. 2C). Moreover, levels of cleaved PARP
and cleaved caspase 3 were noticeably increased by the ERK
inhibitor in the 2 liver cancer cell lines (Fig. 2D). These findings
suggest that DHA-induced apoptosis may be related to ERK
HDACi facilitated DHA-induced apoptosis in liver cancer
In view of that HDACi is capable of enhancing the lethal effect
of chemotherapeutic agents [22,23], we intended to examine
whether DHA combined with HDACi resulted in more cell death
in liver cancer. NaB and SAHA were used in MTT analysis.
According to the results, combination of DHA and HDACi
significantly reduced cell viabilities in liver cancer cells, compared
to treatment with single agent (Fig. 3A). The increased cytotoxicity
of combination of DHA and HDACi was also determined by
colony formation assay. Cells in DMSO groups formed a number
of visible colonies in 15 d. The number of colony formed by cells
cultured with both DHA and HDACi was significantly less than
that with DHA alone (Fig. 3B).
Next we determined the pro-apoptotic activity of combined
treatment with DHA and HDACi. DHA treatment potently
induced apoptosis in liver cancer cells, but more apoptosis was
induced by the combined treatment with both agents, as shown by
TUNEL assays indicating a noticeable increase in
TUNELpositive cells (Fig. 3C). Statistically, DHA in combination with
NaB or SAHA increased apoptotic cells by 1.9 or 2.8 fold,
respectively in Hep G2 cells, and by 3.0 or 3.5 fold respectively in
PLC/PRF/5 cells (Fig. 3D). In line with the increased apoptosis,
caspase 3 activity was higher in cells treated with both DHA and
HDACi (Fig. 3E).
Release of cytochrome c into cytoplasm and
downregulation of Mcl-1 and p-ERK contributed to
apoptosis caused by the combined treatment with
HDACi and DHA
We have previously demonstrated that DHA-induced apoptosis
associated with Mcl-1 degradation and Bak activation . We
next examined whether Mcl-1 and Bak were involved in
HDACimediated enrichment of apoptosis in DHA-treated cells. As
indicated in results of western blot, Mcl-1 was dramatically
decreased, whereas Bak was markedly increased in Hep G2 cells
treated with both DHA and NaB/SAHA. The alterations of Mcl-1
and Bak in PLC/PRF/5 cells shared a similar trend with those in
Hep G2 cells (Fig. 4A). In addition, wild-type p53 in Hep G2 cells
were upregulated, while mutant p53 in PLC/PRF/5 cells were
hardly affected, following the treatment (Fig. 4A).
In light of emerging data that ERK phosphorylation was
inhibited in DHA-treated and HDACi-treated cells. We next
examined the level of phosphorylated ERK. Results showed
a rapid decrease of p-ERK in liver cancer cells treated with both
DHA and SAHA, especially in PLC/PRF/5 cells (Fig. 4B).
Since DHA-induced apoptosis was attributed to the
depolarization of mitochondrial outer membrane , we next examined
Figure 1. Activations of MAP kinases were involved in DHA-induced apoptosis. A. DHA induced apoptosis in liver cancer cells. Cells
treated with either DMSO or 10 mM DHA for 24 and 48 h were stained with both Annexin V and Propidium Iodide (PI) for 45 min. Apoptosis induced
by DHA was then assessed by flow cytometer analysis. B. The percentage of apoptotic cells were shown, after quantitative analysis of PI/Annexin V
assay. Data are presented as mean6SD of three independent experiments. *P,0.05, versus the DMSO group. C. MAP kinases were activated by DHA.
Cells were treated with 10 mM DHA for indicated time. The phosphorylation of p38, ERK and JNK was determined. D. Quantitative data from three
independent experiments were shown to indicate the relative expression of p-p38, p-JNK, and p-ERK.
the reduction of mitochondrial membrane potential (MMP).
Results showed that the combined treatment remarkably lowered
the mitochondrial transmembrane potential (Fig. 4C), followed by
an obvious release of cytochrome c from mitochondria to
cytoplasm (Fig. 4D).
As shown in our previous study, HDACi pretreatment sensitized
liver cancer cells to etoposide . We pretreated cells with NaB
or SAHA, and then assessed the resulting apoptosis by TUNEL
assays. Compared to those of unpretreated cells, percentages of
TUNEL-positive cells in HDACi-pretreated cells were
significantly increased (Fig. 4E).
SAHA enhanced antitumor effect of DHA on Hep G2
xenograft tumor in mice
Having demonstrated the ability of SAHA to enhance
DHAmediated cell death in vitro, we further determined the synergetic
effect of SAHA and DHA in vivo. Hep G2 cells were
subcutaneously injected in nude mice to establish tumor xenograft.
Nude mice bearing tumor xenografts were dosed with DHA
(5 mg/kg/Bid) and/or SAHA (1.5 mg/kg/Bid) daily for 24 days.
The treatment did not appear to have a noticeable effect on body
weight in mice. On average, the combination therapy inhibited
liver cancer tumor growth by more than 44.7% while the single
agent treatment with either DHA or SAHA only inhibited the
tumor growth by 17.6% and 4.6%, respectively (Fig. 5A). On Day
24, mice were sacrificed and the tumor weights were measured. As
expected, the combination of HDACi and DHA significantly
reduced the weights of xenograft tumor, compared with
DHAonly groups (Fig. 5B). These data indicated that the combination
treatment generated a greater anti-proliferative effect and
cytotoxicity than either single agent alone in liver cancer
xenografts in vivo.
In order to test whether HDACi enhanced the lethal effect of
DHA via increasing apoptosis, tumor tissues were sectioned and
subjected to in situ cell death detection (Fig. 5C). Results showed
that the proportion of apoptotic cells was significantly increased
from 8.662.4% in DHA group to 17.763.3% in combined
treatment group (Fig. 5D). In addition, we examined the histology
of tumors after the treatment using H&E staining. Tumors from
control group showed typical histological appearance of liver
cancer (Fig. 6). The sections of DHA-treated tumors showed that
cancer cells were markedly decreased, with signs of apoptosis,
infiltration of inflammatory cells and fibrosis. In the combined
treated group, apoptotic regions and extensive necrosis with
infiltration of phagocytic cells could be observed fairly often.
In addition, we performed immunohistochemistry to detect the
proteins involved in HDACi/DHA-induced apoptosis (Fig. 6).
Decreased expression of Ki-67 indicated the reduction of cell
proliferation and likely enhanced cell death. Detectable difference
in p53 expression was observed. Striking increase of active PARP,
as well as a predominant decline of Mcl-1 and p-ERK, was present
in HDACi/DHA-treated xenograft. Taken together, these data
indicated that HDACi were able to significantly augment
DHAmediated antitumor effects.
Recent studies suggest that HDACi including NaB and SAHA
interact synergistically with cytotoxic agents, such as fludarabine
and etoposide, to dramatically increase mitochondrial injury and
apoptosis in leukemic and epithelial cancer cells [24,25]. The
antitumorigenic properties of HDACi are especially notable
probably due to the fact that their cytotoxic effects are usually
specific to cancer cells but not to normal cells. However, when
used as a single agent, HDACi might exhibit limited lethal activity
towards liver cancer, which is evident in the present study showing
that both NaB and SAHA at low doses are unable to induce
significant growth inhibition in vitro and in vivo. However, when
HDACi are used in combination with DHA, a derivative of
artemisinin that is clinically used in malaria treatment with good
toxicity profile , they can induce much more apoptosis,
resulting in remarkable halt of tumor xenograft in nude mice.
There is very limited information on HDACi in combination with
other anti-tumor agents against liver cancer. Our data for the first
time have demonstrated a synergic effect of DHA and HDACi in
inhibition of liver cancer.
Many reports have demonstrated that the threshold of apoptosis
in cancer cells can be controlled by the activities of multiple signal
transduction pathways, one of which is Raf-MEK1/2-ERK1/2
pathway [27,28]. This pathway is frequently dysregulated in
neoplastic transformation, along with the c-Jun NH2-terminal
kinase (JNK1/2) and p38 MAPK pathways . It has also been
implicated that activation of the ERK1/2 pathway is usually
associated with survival but JNK1/2 and p38 MAPK pathway
with apoptosis . In our study, ERK1/2 phosphorylation was
slightly inhibited by DHA treatment but strongly inhibited by the
combined treatment with DHA and HDACi. In addition, using
the ERK-specific inhibitor PD98059, we demonstrated that the
activation of the ERK is antiapoptotic since the ERK inhibitor
enhanced DHA-induced apoptosis in liver cancer cells.
A number of antiapoptotic effector proteins have been identified
downstream of ERK1/2 signaling, including Bcl-xL and Mcl-1
[31,32]. Alterations of both phosphorylated ERK and Mcl-1
frequently occurs in the same direction. Yuen et al. reported that
silencing Ran may lead to deactivation of ERK and
downregulation of Mcl-1 in cancer cells . Reeves et al. showed that
the activation of ERK and induction of Mcl-1 were observed in
myeloid cells infected by human cytomegalovirus . Calvin o
et al. reported treatement with lonidamine plus arsenic trioxide
resulted in reductions of Mcl-1 and p-ERK . In our previous
study, Mcl-1 was downregulated in DHA-treated cells [ref]. Here
we further showed a decrease of phosphorylated ERK in
DHAexposed liver cancer cells. Collectively, it seems that there is some
certain correction between Mcl-1 and p-ERK: one protein could
be regulated by the other. Interestingly, Konopleva et al. showed
that MEK inhibitors such as PD0325901 and CI-1040, which are
capable of inhibiting the activation of ERK, successfully
suppressed Mcl-1 expression in Leukemia cells . Booy et al.
demonstrated that knockdown of ERK1 or inhibition of the ERK
phosphorylation sufficiently inhibited EGF-mediated Mcl-1
upregulation . Sun et al. showed that the overexpression of ERK
partly reversed EPOX-induced Mcl-1 degradation in tumor cells
. However, the detailed mechanism through which ERK
affects Mcl-1 expression requires further investigation. In view of
that (a) a decrease of Mcl-1 is essential for the induction of
apoptosis by diverse apoptotic stimuli caused by different types of
chemotherapeutic agents; (b) deactivation of ERK may result in
Mcl-1 degradation; and (c) the administration of both HDACi and
DHA synergistically regulate ERK phosphorylation and Mcl-1
Figure 6. Decreased expression of Mcl-1 and increased levels of active PARP and cleaved caspase 3 were recorded in
HDACi/DHAtreated mice. SAHA enlarged the apoptotic region caused by DHA treatment in Hep G2 xenograft tumor. Tumors were excised and subjected to
H&E staining for determination of pathological evaluation. On the other hand, tissues of xenografts were subjected to immunochemistry to detect
the expression of Ki-67, p53, Mcl-1, p-ERK, and active PARP. Original magnification 6400.
expression, the combination treatment with HDACi and DHA
should have a great clinical potential in the improvement of liver
In light of the findings that (a) DHA induced more cell death in
liver cancer cells bearing wild-type p53, (b) HDACi can lead to
upregulation of p53, we rationally assumed that the combined
treatment with DHA and HDACi increased apoptosis probably
via inducing p53 expression in Hep G2 cells. However, more
evidence should be obtained to verify the assumption. On the
other hand, cells with p53 mutants have been demonstrated to be
less sensitive to DHA, but can significantly respond to HDACi
treatment . Therefore, if the combination of both agents is
used to treat p53-mutated cells, the apoptosis induced is likely to
be comparable with that in cells with wild-type p53. In fact, such
an assumption is proved in our present experiment. This finding
indicates that HDACi and DHA in combination can be applied to
both p53-wide type and p53-mutated liver cancer with similar
efficacy. This is of clinical significance since p53 mutants are
presented in most of liver cancer cases.
According to our results that (a) in vitro data showed that
combination of HDACi and DHA significantly reduced cell
viability in both cells, and (b) in vivo data revealed a remarkable
decrease of Ki-67, this combined treatment resulted in significant
cell growth inhibition which may lead to the resulting antitumor
effects. However, further study should be carried on to disclose the
exact mechanism through which cell growth inhibition, in
addiction to apoptosis, caused by combination of HDACi and
DHA contributed to tumor inhibition.
In conclusion, our in vitro and in vivo data highlight that the
combination treatment with HDACi and DHA has a synergic
effect in the induction of liver cancer cell death, and this strategy
can also reduce the dose of HDACi and thus it may circumvent
the inherent toxicity. Mechanically, our study has demonstrated
that the combination treatment can regulate ERK
phosphorylation and Mcl-1 expression to induce apoptosis of liver cancer cells
independent of p53 status.
Conceived and designed the experiments: JPY GGC CZYZ. Performed
the experiments: CZYZ YHP YC LLL. Analyzed the data: JPY GGC
CZYZ PBL. Wrote the paper: CZYZ YHP YC PBL JPY.
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