Progesterone Increases Apoptosis and Inversely Decreases

Hindawi Publishing Corporation
e Scientific World Journal
Volume 2014, Article ID 567148, 11 pages
http://dx.doi.org/10.1155/2014/567148
Research Article
Progesterone Increases Apoptosis and Inversely
Decreases Autophagy in Human Hepatoma HA22T/VGH
Cells Treated with Epirubicin
Wen-Tsan Chang,1,2,3 Hsiao-Ling Cheng,4 Bau-Shan Hsieh,4
Chien-Chih Chiu,5 King-Teh Lee,1,2,3 and Kee-Lung Chang1,4
1
Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan
Division of Hepatobiliary and Pancreatic Surgery, Department of Surgery, Kaohsiung Medical University Hospital,
Kaohsiung 80756, Taiwan
3
Department of Surgery, School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan
4
Department of Biochemistry, School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan
5
Department of Biotechnology, College of Life Science, Kaohsiung Medical University, Kaohsiung 80708, Taiwan
2
Correspondence should be addressed to King-Teh Lee; [email protected] and Kee-Lung Chang; [email protected]
Received 20 March 2014; Accepted 1 May 2014; Published 19 May 2014
Academic Editor: Hsueh-Wei Chang
Copyright © 2014 Wen-Tsan Chang et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
Hepatocellular carcinoma (HCC) is the leading cause of cancer-related deaths worldwide. Epirubicin can induce intracellular
reactive oxygen species and is widely used to treat unresectable HCC. Progesterone has been found to inhibit the proliferation
of hepatoma cells. This study was designed to test the combined effects of epirubicin and progesterone on human hepatoma
cell line, HA22T/VGH. These cells were treated with different concentrations of epirubicin with or without the coaddition of
30 M progesterone and then analyzed for apoptosis, autophagy, and expressions of apoptotic-related proteins and multidrugresistant gene. Epirubicin treatment dose-dependently inhibited the growth of HA22T/VGH cells. Addition of 30 M progesterone,
which was inactive alone, augmented the effect of epirubicin on the inhibition of growth of HA22T/VGH cells. Cotreatment with
progesterone enhanced epirubicin-induced apoptosis, as evidenced by greater increase in caspase-3 activity and in the ratio of the
apoptosis-regulating protein, Bax/Bcl-XL . The combination also caused a decrease in autophagy and in the expression of multidrug
resistance-related protein 1 mRNA compared to epirubicin alone. This study shows the epirubicin/progesterone combination was
more effective in increasing apoptosis and inversely decreasing autophagy on HA22T/VGH cells treated with epirubicin alone,
suggesting that this combination can potentially be used to treat HCC.
1. Introduction
Hepatocellular carcinoma (HCC) is the fifth most common
cancer in men and the seventh in women, and it is the third
most common cause of cancer-related deaths worldwide [1,
2]. Liver resection, local ablation therapy, and liver transplantation are the suggested curative therapies for HCC, while
transarterial chemoembolization (TACE) has been used to
treat unresectable HCC with some clinical efficacy [3–5].
Anthracyclines, such as doxorubicin or epirubicin, have
been widely used to treat advanced HCC, to prevent or
treat postoperative recurrence, and to downstage the disease before liver transplantation by systemic infusion or
by transarterial route [6, 7]. Acting as topoisomerase-II
inhibitors, anthracycline drugs induce DNA damages and
acute oxidative stress in cells [8, 9]. And the quinine group of
anthracyclines can cause one electron reduction to produce
a semiquinone [9, 10]. The free radical semiquinone consequently produces reactive oxygen species (ROS), including
superoxide anions, hydrogen peroxide, and hydroxyl radicals
[9, 10]. ROS are reported to be involved in epirubicin-induced
apoptosis in hepatoma cell lines [10]. Furthermore, epirubicin
2
is less cardiotoxic than doxorubicin [6]. Therefore, epirubicin
is widely used in Europe and Asia in the treatments of cancers,
including HCC [6, 8].
However, the side effects of anthracyclines include cardiomyopathy, immunosuppression, and the development of
primary or secondary drug resistance, which may sometimes
adversely affect survival, recurrence, and extrahepatic metastases in HCC patients [11–13]. And HCC cells themselves are
usually resistant to chemotherapeutic agents, the response
rates of chemotherapy in HCC are reported to be only 10.5%–
20.6%, and, generally, overall survivals have been less than 12
months for advanced HCC patients [6, 12].
Autophagy, a survival mechanism of some cancer cells,
can be induced during starvation, chemotherapy, radiation,
hypoxia, and some endocrine therapies [14, 15]. Sun et al
reported that autophagy could protect breast cancer cells
from epirubicin-induced apoptosis and facilitate the development of epirubicin resistance [16]. Progesterone can reverse
multidrug resistance gene expression in epirubicin-treated
urethral cancer cell lines via p-glycoprotein pathway [17].
And megestrol (a progestin drug) treatment has produced
some efficacy in advanced HCC in some clinical studies
[18, 19]. Therefore, combining epirubicin with progesterone
might be a potential strategy for treating HCC, as it might
allow for the smaller doses of epirubicin, which is generally
toxic, while increasing its effectiveness against HCC and
decreasing epirubicin-related side effects.
Our previous study showed HA22T/VGH cells are susceptible to changes in redox status and oxidative stresses also
induce apoptosis in these cells [20]. This study tested the
effect of combining epirubicin with progesterone to treat a
metastatic, poorly differentiated HCC cell line, HA22T/VGH.
To evaluate the therapeutic effect of this combination, we
analyzed occurrence of apoptosis and autophagy, expressions
of their related proteins, and the expression of multidrug
resistance-related protein 1 (MRP-1) gene.
2. Materials and Methods
2.1. Reagents and Antibodies. Epirubicin hydrochloride
(Pharmorubicin) was purchased from Pfizer Italia S.R.L.
(Milano, Italy), progesterone from Sigma-Aldrich (St.
Louis, MO, USA), acridine orange from Molecular Probes
(Eugene, OR, USA), protein assay reagents from Bio-Rad
Laboratories (Hercules, CA, USA), and TRIzol reagent from
Invitrogen Life Technologies (Carlsbad, CA, USA). All other
chemicals were of analytical grade and purchased from
Sigma-Aldrich (St. Louis, MO, USA). Mouse monoclonal
antibodies against light chain-3 (LC-3), Beclin-1, or Bax,
rabbit polyclonal antibodies against Bcl-XL , and goat
polyclonal antibodies against -actin were purchased
from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Horseradish peroxidase-conjugated anti-mouse, -goat, and
-rabbit IgG antibodies were purchased from BD Pharmingen
Inc. (San Diego, CA, USA).
2.2. Cell Line, Cell Culture, and Drug Treatments. HA22T/
VGH cell line was obtained from the Food Industry Research
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and Development Institute in Hsinchu, Taiwan (BCRC number: 60168) and was cultured in Dulbecco’s modified Eagle’s
medium (DMEM) (Gibco BRL, Grand Island, NY, USA)
containing 10% fetal bovine serum (FBS) (Hyclone, Auckland, NZ), 2 mM L-glutamine (Gibco BRL, Grand Island, NY,
USA), 0.1 mM nonessential amino acids (Gibco BRL, Grand
Island, NY, USA), 100 units/mL of penicillin, and 100 g/mL
of streptomycin (Gibco BRL, Grand Island, NY, USA) at 37∘ C
in a humidified chamber with 5% CO2 . To investigate the
effects of epirubicin and progesterone, various concentrations
of epirubicin and progesterone were added to the culture
medium for an indicated time period and then the cells were
harvested and analyzed.
2.3. Cell Growth. After epirubicin and progesterone treatment, the cells were harvested and viable cells were counted
using a dye exclusion technique as described previously [21].
Briefly, the cell suspension was centrifuged at 5,000 ×g; the
supernatant was discarded, and the cell pellet was resuspended in serum-free medium. One volume of 0.4% Trypan
blue (Gibco BRL, Grand Island, NY, USA) was added to one
volume of cell suspension, and then cells were counted in
a hemocytometer after incubation at room temperature for
3 min. All counts were done in triplicate.
2.4. TUNEL Assay. Terminal deoxynucleotidyl transferasemediated dUTP nick-end labeling (TUNEL) assays were performed using an APO-BrdU TUNEL Assay Kit (Molecular
Probes, Eugene, OR, USA) according to the manufacturer’s
directions as described previously [21]. Briefly, the cells were
incubated for the indicated time before being trypsinized,
washed with phosphate-buffered saline (PBS), and fixed in
2% paraformaldehyde (pH 7.4) for 15 min. The fixed cells
were washed twice in PBS and stored at −20∘ C in 70%
ethanol for 12–18 h prior to performing the TUNEL assay.
After removing the 70% ethanol by centrifugation, the cells
were washed twice in wash buffer and then incubated at 37∘ C
for 60 min with DNA-labeling solution containing terminal
deoxynucleotidyl transferase and BrdUTP. After washing
twice with rinse buffer, the cells were resuspended for
30 min in the dark at room temperature in antibody solution
containing Alexa Fluor 488-labeled anti-BrdU antibody. Flow
cytometric analysis was subsequently performed using a
Coulter Epics XL cytometer (Beckman Coulter, Miami, FL,
USA) to quantify the fluorescence intensity for determination
of apoptotic status. The data were analyzed using WINMDI
software version 2.8 (Scripps Research Institute, La Jolla, CA,
USA), with a minimum of 1 × 104 cells per sample being
evaluated in each case.
2.5. Caspase-3 Colorimetric Protease Assay. The activity of
caspase-3 was detected using an ApoTarget caspase-3 colorimetric protease assay kit (Invitrogen Corp., Camarillo,
CA, USA) according to the manufacturer’s instructions as
described previously [21]. Briefly, we induced apoptosis in
cells by epirubicin and/or progesterone treatments while
concurrently incubating a control culture without induction.
We then counted cells as pellet 3–5×106 cells per sample.
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The cells were resuspended in 50 L of chilled Cell Lysis
Buffer, incubated on ice for 10 min, and then centrifuged for
1 min in a microcentrifuge (10000 ×g). Supernatant (cytosol
extract) was transferred to a fresh tube and put on ice and
protein assay reagents (Bio-Rad Laboratories, Hercules, CA,
USA) were used. Each cytosol extract was diluted to a concentration of 50–200 g protein per 50 L Cell Lysis Buffer (1–
4 mg/mL). A 50 L of 2 × reaction buffer (containing 10 mM
DTT) was added to each sample followed by 5 L of the 4 mM
DEVD-pNA substrate (200 M of final concentration). The
samples were incubated in the dark at 37∘ C for 2 h. Samples
were read in a microplate reader set at 405 nm. Fold increase
in caspase-3 activity was determined compared to that in
untreated controls.
2.6. Detection of Autophagy with Acridine Orange Staining.
Formation of acidic vesicular organelles (AVOs), a morphological characteristic of autophagy, was quantified by acridine
orange staining as described previously [22]. In brief, acridine
orange (1 g/mL) was added 30 min prior to collection, and
after being washed with PBS, cells were analyzed using the
Coulter Epics XL cytometer (Beckman Coulter, Miami, FL,
USA). Green (510–530 nm) and red (>650 nm) fluorescence
emission from 1 × 104 cells illuminated with blue (488 nm)
excitation light was measured. The data were analyzed using
WINMDI software version 2.8 (Scripps Research Institute, La
Jolla, CA, USA), with a minimum of 1 × 104 cells per sample
being evaluated in each case.
2.7. Western Blotting. Sample preparation and Western blotting procedures were performed as described previously
[21]. Briefly, cells were harvested and cytosolic extracts were
prepared using lysis buffer (20 mM Tris-HCl (pH 7.2), 2 mM
EGTA, 5 mM EDTA, 500 M sodium orthovanadate, 10 mM
sodium fluoride, 1% Triton X-100, 0.1% SDS, and protease
inhibitor cocktail). Protein concentrations were determined
using protein assay reagents. Forty to sixty micrograms
of protein lysate was analyzed by SDS-polyacrylamide gel
electrophoresis. After transfer of the proteins from the gel to
a nitrocellulose membrane (Amersham Pharmacia Biotech,
Freiburg, Germany), the membranes were blocked for 1 h
at room temperature in PBS with 0.05% Tween 20 (PBS-T)
containing 5% nonfat dry milk, and then they were incubated
with specific primary antibodies and horseradish peroxidaseconjugated secondary antibodies. The immunoreactive bands
were visualized using an enhanced chemiluminescence kit
(Perkin-Elmer Life Sciences, Boston, MA, USA).
2.8. Reverse Transcription-Polymerase Chain Reaction (RTPCR). Total RNA was extracted from cells with TRIzol
reagent (Invitrogen Life Technologies, CA, USA) according
to the manufacturer’s instructions as described previously
[23]. The complementary DNA (cDNA) was synthesized
from random hexadeoxynucleotide primed reverse transcription from 2 g of total RNA using M-MLV reverse transcriptase (Promega Corporation, WI, USA) according to the
manufacturer’s directions. Polymerase chain reaction (PCR)
was then performed using the Dream Taq DNA polymerase
3
(Thermo scientific, MA, USA) on an Applied Biosystems
Gene Amp9700 PCR system (Applied Biosystems, Foster,
CA, USA). The thermocycling began with 94∘ C for 5 min followed by 30 cycles of 94∘ C for 1 min, 60∘ C for 1 min, and 72∘ C
for 1 min, and then followed by 70∘ C for 10 min. PCR primers
sequences were as follows: multidrug resistance-related protein 1 (MRP-1) forward, 5 -AGG TGGACCTGT TTC GTG
AC-3 ; reverse, 5 -ACCCTGTGATCCACCAGAAG-3 , and
GAPDH forward, 5 -GAC ATC AAG AAG GTG GTG AAG
CAG-3 ; reverse, 5 -GCG TCA AAG GTG GAG GAG TGG3 . The amplified PCR products were analyzed on 2% agarose
gels and photographs were taken. The intensity of each
band was calculated by densitometry analysis and the results
were expressed as a percentage of the optical density of the
corresponding GAPDH band.
2.9. Statistical Analysis. Comparisons among the groups of
cells, one-way analysis of variance (ANOVA), and Fisher’s
least significant difference test were performed using the SPSS
17.0 statistical software (SPSS, Chicago, IL). All experiments
were performed at least thrice. All data are expressed as the
mean ± standard deviation (S.D.). Value differences were
considered significant if  < 0.05.
3. Results and Discussions
3.1. Inhibition of Cell Growth. To gain initial insight into the
effects of epirubicin alone or in combination with progesterone on cell growth of hepatoma cell line, HA22T/VGH
cells were treated for 24 or 48 h without or with different
doses of epirubicin in the absence or presence of 30 M
progesterone. The IC50 of progesterone was 100 M and
almost not cytotoxic to HA22T/VGH cells at concentrations
<50 M (data not shown). Therefore, a concentration of
30 M progesterone was the dosage chosen for the cotreatment with epirubicin. During the 24 h incubation period, the
untreated HA22T/VGH cells proliferated, while the growth
of the cells treated with epirubicin ≥0.3 M was significantly
inhibited. The addition of 30 M progesterone significantly
augmented epirubicin’s inhibition of growth at concentration
of ≥0.1 M (Figure 1(a)). During 48 h incubation, epirubicin
inhibited cell growth in a dose-dependent manner and the
coaddition of progesterone augmented its effect (Figure 1(b)).
3.2. Induction of Apoptosis and Expressions of ApoptosisRelated Proteins. Apoptotic cells were measured by flow
cytometric analysis after TUNEL staining (Figure 2(a)). After
24 h treatment with epirubicin, a significant increase in the
percentage of TUNEL-positive apoptotic cells was seen as
compared with controls at concentrations ≥0.1 M epirubicin. Although progesterone alone did not cause a significant change in the number of apoptotic cells, using it in
combination with epirubicin at concentrations ≥0.3 M had
a stronger effect. The fluorescence intensities of apoptotic
cells of HA22T/VGH cells treated with 30 M progesterone,
0.3 M epirubicin, or combination therapy were 102.2 ±
20.3%, 193.8 ± 20.0%, and 264.0 ± 38.0%, respectively, compared with those of controls (Figure 2(a)). Figure 2(b) shows
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(b)
Figure 1: Effects of epirubicin on cell growth without or with progesterone addition. HA22T/VGH cells were treated with different
concentrations of epirubicin without or with 30 M progesterone for 24 h (a) or 48 h (b). Results are expressed as the mean ± standard
deviation (S.D.) for three separate experiments. E: epirubicin; P30: 30 M progesterone. ∗  < 0.05 compared to the corresponding untreated
controls. #  < 0.05 compared to the corresponding epirubicin-treated group.
that adding 30 M progesterone to 0.3 M epirubicin treatment produced more caspase-3 activity (212.5 ± 10.6%) than
using 0.3 M epirubicin alone (126.0 ± 5.7%). These results
showed epirubicin at concentration of 0.3 M activated
caspase-3 and induced apoptosis, and combining the two
drugs had a significant effect on apoptosis in HA22T/VGH
cells.
To determine whether the treatment-induced apoptosis
was associated with altered expression of apoptosis-relating
proteins, HA22T/VGH cells were treated for 24 h with 0.3 M
epirubicin in the presence or absence of 30 M progesterone and analyzed by Western blotting. Figure 3 shows that
progesterone alone had no significant effect (94.8 ± 10.6%
versus 100 ± 0.0%) on the antiapoptotic protein, Bcl-XL
levels, whereas epirubicin caused a decrease (71.1 ± 13.6%
versus 100 ± 0.0%;  < 0.05), compared with those of
controls. The combination of epirubicin and progesterone
caused a marked decrease in expression of Bcl-XL compared
to 0.3 M epirubicin alone (13.9 ± 4.8% versus 71.1 ± 13.6;  <
0.05). Meanwhile, progesterone alone had no effect on the
proapoptotic protein, Bax levels; epirubicin alone decreased
Bax levels. The coaddition of progesterone and epirubicin
significantly lessened the decrease of Bax levels compared to
epirubicin alone (84.7 ± 31.3% versus 38.0 ± 7.1%;  < 0.05).
Thus, the ratio of proapoptotic/antiapoptotic factor, Bax/BclXL , was extremely enhanced by the combination therapy,
which can partly explain why apoptosis was increased by the
combination (Figure 3).
Activation of caspase-3 is an essential step in apoptosis.
Our results demonstrated progesterone augmented caspase-3
activity of epirubicin-treated HA22T/VGH cells significantly.
Epirubicin or doxorubicin can induce intracellular ROS
[8, 9]. ROS production may interact with Fas-associated
death domain (FADD) pathway and FADD sequence can
result in activation of caspase-3 which has been reported
in various cancer cell lines [24, 25]. This study also found
that progesterone interfered with the expression of apoptosisregulating proteins, upregulating Bax and downregulating
Bcl-XL , in the epirubicin-treated HA22T/VGH cells. It is
currently unknown whether progesterone initially triggers
apoptosis upstream from caspase-3 or not. Bcl-XL expression
is important for the inhibition of apoptosis initiated by
various cellular stresses in human HCC cells [26, 27]. We,
therefore, propose that the Bcl-2 family may contribute to
the improved efficacy of treating HA22T/VGH cells with a
combination of epirubicin and progesterone. On the other
hand, the expression of the progesterone receptor and its
potential role in HA22T/VGH cells have not been reported
till now; however, some studies have evaluated the role of the
progesterone receptor-mediated apoptosis in other human
hepatoma cells. Cheng et al. demonstrated that treatment
with RU486, a progesterone receptor antagonist, inhibits the
progesterone-mediated response to estradiol pretreatment in
tumor necrosis factor-induced apoptotic Huh-7 cells [28]. On
the contrary, Zhang and Chow reported that the progesterone
receptor is not involved in the action of megestrol-induced
apoptosis in HepG2 cells [29]. Thus, further studies on the
potential role of the progesterone receptor in HA2T/VGH
cells are necessary.
3.3. Autophagy Reduction by Combination. It has been
reported that autophagy can be induced during chemotherapy [30, 31]. To determine whether the treatments had
an effect on autophagy induction, HA22T/VGH cells were
treated for 24 h with 0.3 M epirubicin in the presence or
absence of 30 M progesterone, then subjected to acridine
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TUNEL-positive cells (% of control)
400
(b)
Figure 2: Apoptosis induction by epirubicin without or with progesterone addition. HA22T/VGH cells were treated with indicated
concentrations of epirubicin without or with 30 M progesterone for 24 h and evaluated by (a) TUNEL staining or (b) caspase-3 activity.
Results are expressed as the mean ± standard deviation (S.D.) for three separate experiments. C: untreated cells, E0.03: 0.03 M epirubicin,
E0.1: 0.1 M epirubicin, E0.3: 0.3 M epirubicin, and P30: 30 M progesterone. ∗  < 0.05 compared to the untreated controls. #  < 0.05
compared to the corresponding epirubicin-treated group.
orange staining, and analyzed by flow cytometry. Figure 4
shows both epirubicin and progesterone increased autophagy
compared to controls by AVOs analysis, though epirubicin
was more effective than progesterone. Surprisingly, coaddition of progesterone significantly reduced the epirubicininduced increase of autophagy. To further explore the expression of autophagy-related proteins, HA22T/VGH cells were
treated for 24 h with 0.3 M epirubicin in the presence
or absence of 30 M progesterone and then subjected to
Western blotting. As shown in Figure 5, neither epirubicin
nor progesterone had an effect on Beclin-1 levels, but the
combination of the two significantly reduced Beclin-1 levels.
The expressions of proteins of Beclin-1 of HA22T/VGH cells
treated with 0.3 M epirubicin, 30 M progesterone, or combination therapy for 24 h were 119.9 ± 19.5%, 113.6 ± 1.4%, and
77.4 ± 2.6%, respectively, compared with those of controls.
Figure 5 also shows progesterone had no effect on LC3-I
levels, whereas epirubicin markedly reduced LC3-I levels,
indicating that it may convert LC3-I to LC3-II. Interestingly,
coaddition of progesterone to epirubicin treatment significantly reversed LC3-I levels. The expressions of proteins of
LC3-I of HA22T/VGH cells treated with 0.3 M epirubicin,
30 M progesterone, or combination therapy for 24h were
21.5 ± 16.2%, 76.6 ± 17.3%, and 99.8 ± 25.6%, respectively,
compared with those of controls. This is compatible with the
results of AVOs formation shown in Figure 4.
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Figure 3: Western blotting shows Bax and Bcl-XL expressions of HA22T/VGH cells after epirubicin and/or progesterone treatment for 24 h.
Results are expressed as the mean ± standard deviation (S.D.) for three separate experiments. C: untreated cells, E0.3: 0.3 M epirubicin, and
P30: 30 M progesterone. ∗  < 0.05 compared to the untreated controls. #  < 0.05 compared to epirubicin-treated group.
Many studies have indicated that autophagy can serve
as a survival mechanism for cancer cells and suggested that
autophagy inhibitor might enhance the antitumor effects
of chemotherapy or target therapy agents in vivo [30, 31].
In addition, Shen et al. reported inhibition of autophagy
could enhance proapoptotic effects of ZD6474 in glioblastoma cells [31]. Greene et al. also reported inhibition of
late-stage autophagy synergistically enhanced pyrrolo-1, 5benzoxazepine-6-induced apoptotic cell death in human
colon cancer cells [32]. In this study, progesterone was
found to be able to reduce epirubicin-induced autophagy
in HA22T/VGH cells. There are some interactions between
autophagy and apoptosis mediated by Beclin-1 and Bcl-XL
proteins [32–34]. Beclin-1 is an autophagy-related protein,
while Bcl-XL is an anti-apoptosis-related protein. However, Bcl-XL and Bcl-2 have been reported to be negative
regulators of Beclin-1 [33–35]. Bcl-XL can inhibit Beclin1 activity by stabilizing Beclin-1 homodimerization [34].
Akar et al. reported that doxorubicin induced autophagy
through the upregulation of Beclin-1, which was further
enhanced by siRNA-mediated Bcl-2 silencing MCF-7 cells
[36]. The expressions of Beclin-1 and AVOs were compatible
in Figures 4 and 5, indicating that 30 M progesterone
could decrease the expression of AVOs in HA22T/VGH cells
treated with 0.3 M epirubicin and these effects may be
induced by suppression of expression of Beclin-1. In contrast,
the present study found that after progesterone was added,
the expressions of Bcl-XL and Beclin-1 both were reduced.
Our results proved that progesterone in combination with
epirubicin could increase the epirubicin-induced apoptosis
and decrease epirubicin-induced autophagy in HA22T/VGH
cells. Therefore, the decreased expression of Beclin-1 could
not be explained by the interactions between Beclin-1 and
Bcl-XL described above.
Thus, Figure 4 displays progesterone increased autophagy
compared to controls maybe due to the fact that 30 M
progesterone does not augment the expression of BclXL shown in Figure 3. It has been demonstrated that
101
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P30
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634.8
± 72.1
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0
Figure 4: Epirubicin and/or progesterone effect on autophagy induction. HA22T/VGH cells were treated with epirubicin and/or progesterone
for 24 h then stained with acridine orange and followed by flow cytometric analysis of autophagy. Results are expressed as the mean ± standard
deviation (S.D.) for three separate experiments. C: untreated cells, E0.03: 0.03 M epirubicin, E0.1: 0.1 M epirubicin, E0.3: 0.3 M epirubicin,
and P30: 30 M progesterone. ∗  < 0.05 compared to the untreated controls. #  < 0.05 compared to epirubicin-treated group.
the Toll-like receptor 4 (TLR4)—myeloid differentiation
factor 88 (MyD88) pathway can mediate lipopolysaccharide (LPS)—induced autophagy by reducing the binding
of Beclin-1 and Bcl-2 and thus triggers autophagy activation in human and murine macrophages [37, 38]. Su et
al. reported progesterone inhibited TLR4-mediated innate
immune response in murine macrophages [39]. Hepatocytes
also express TLR4 receptors and are responsive to LPS
[38, 40]. Our preliminary study also shows combination of
progesterone and epirubicin can decrease epirubicin-induced
expression of TLR4 and MyD88 and sequent production
of interlukin-6 in HA22T/VGH cells (unpublished data).
Thus, coaddition of progesterone significantly reduced the
epirubicin-induced autophagy in HA22T/VGH cells which
may be caused by inhibition of TLR4-MyD88 pathway by
progesterone. However, further studies on the potential role
of TLR4 and MyD88 pathway in HA22T/VGH cells are
necessary.
The microtubule-associated protein 1-light chain-3
(LC3) is an ubiquitin-like molecule which is a mammalian
homologue of the autophagy-related Atg8 encoded product
in yeast [41]. During the fusion of autophagosomal
membranes, cytosolic LC3 (LC3-I) is conjugated to
phosphatidylethanolamine (PE) through two consecutive
ubiquitylation-like reactions catalyzed by the E1-like
enzyme (Atg7) and E2-like enzyme (Atg3) to form the LC3phospholipid conjugate (LC3-II) [42, 43]. During the fusion
of autophagosomes with lysosomes, intra-autophagosomal
LC3-II is also degraded by lysosomal proteases [41, 43].
In this study, we found epirubicin alone enhanced
8
The Scientific World Journal
C
E0.3
P30
E0.3 + P30
Beclin-1
LC3-I
Actin
160
160
140
140
120
120
40
60
0
C
0
E0.3 + P30
20
P30
20
E0.3
∗
40
E0.3 + P30
60
80
P30
80
100
E0.3
#
∗
LC3-I (% of control)
100
C
Beclin-1 (% of control)
#
Figure 5: Western blotting shows Beclin-1 and LC3-I expressions in HA22T/VGH cells after epirubicin and/or progesterone treatment for
24 h. Results are expressed as the mean ± standard deviation (S.D.) for three separate experiments. C: untreated cells, E0.3: 0.3 M epirubicin,
and P30: 30 M progesterone. ∗  < 0.05 compared to the untreated controls. #  < 0.05 compared to epirubicin-treated group.
the formation of AVOs but decreased the expression of
LC3-I, whereas coaddition of progesterone decreased the
formation of AVOs and reversed the expression of LC3-I.
These results indicate that epirubicin may promote the
turnover of LC3-I to LC3-II, a possibility that is compatible
with epirubicin-induced formation of AVOs during the same
incubation period (24 h). And addition of progesterone to
epirubicin to treat HA22T/VGH cells significantly decreased
epirubicin-induced autophagy.
3.4. Decrease of Multidrug Resistance-Related Protein 1 (MRP1) mRNA Expression by Combination. We examined the
MRP-1 mRNA expression in 0.3 M epirubicin and/or 30 M
progesterone treated HA22T/VGH cells for 6, 12, or 24 h by
RT-PCR analysis. As Figure 6 shows, there was no significant
difference in MRP-1 mRNA expression by epirubicin and/or
progesterone treatment for 6 h or 12 h, whereas cotreatment of epirubicin and progesterone produced lower MRP1 mRNA expression after 24 h treatment, suggesting that the
combination might lessen drug resistance in HA22T/VGH
cells. The expressions of MRP-1 mRNA HA22T/VGH cells
treated with 0.3 M epirubicin, 30 M progesterone, or combination therapy for 24 h were 120.1 ± 19.0%, 109.6 ± 21.0%,
and 69.0 ± 12.0%, respectively, compared with those of controls. There are many studies indicating that chemotherapy
can evoke drug resistance and that this resistance may be
related to the expression of multidrug resistance-related protein gene, MRP-1 [44, 45]. Expression of multidrug resistance
protein 1 (MRP-1) has been commonly observed in liver tissue
and HCC cell lines treated with doxorubicin [46, 47]. It
also has been reported that enhanced autophagy can induce
drug-resistance in epirubicin-treated breast cancer cells [16]
and increase of autophagy can induce production of MRP-1
[48]. This study found that the addition of progesterone to
epirubicin-treated HA22T/VGH cells significantly decreased
the expression of multidrug resistance-related protein 1
(MRP-1) gene, a decrease that might be related to the
reduction of autophagy. Because the mechanisms underlying
this possibility are not fully clarified in this study, more in
vitro and in vivo studies are required.
4. Conclusions
Epirubicin is an anthracycline drug that can induce intracellular ROS [8, 9]. This study showed that epirubicin treatment inhibited the growth of HA22T/VGH cells in a dosedependent manner. The addition of 30 M progesterone,
which was inactive by itself, augmented epirubicin’s inhibition of growth of cancer cells. Cotreatment with progesterone
resulted in enhancement of the epirubicin-induced apoptosis,
The Scientific World Journal
9
6h
M
12 h
E0.3
E0.3
+
+
C E0.3 P30 P30 C E0.3 P30 P30
24 h
E0.3
+
C E0.3 P30 P30
combination is worth further evaluation. However, more in
vitro and in vivo studies are required.
Abbreviations
MRP-1
HCC:
AVOs:
FADD:
MRP-1:
ROS:
LC-3:
TLR-4:
MyD88:
GAPDH
200
MRP-1 mRNA (% of control)
180
Hepatocellular carcinoma
Acidic vesicular organelles
Fas-associated death domain
Multidrug resistance-related protein 1
Reactive oxygen species
Light chain-3
Toll-like receptor 4
Myeloid differentiation factor 88.
160
140
Conflict of Interests
120
100
#
∗
80
60
The authors declare that there is no conflict of interests
regarding the publication of this paper.
Authors’ Contribution
40
20
E0.3 + P30
P30
E0.3
C
0
6h
12 h
24 h
Figure 6: Epirubicin and/or progesterone effect on MRP-1 expression. HA22T/VGH cells were treated with epirubicin and/or progesterone for 6, 12, or 24 h, and then MRP-1 mRNA was analyzed by
RT-PCR. The MRP-1 mRNA expression was normalized to GAPDH
mRNA. The density of band was expressed as the relative density
compared to that in untreated cells (control), taken as 100%. Results
are expressed as the mean ± standard deviation (S.D.) for three
separate experiments. C: untreated cells, E0.3: 0.3 M epirubicin,
and P30: 30 M progesterone. ∗  < 0.05 compared to the untreated
controls. #  < 0.05 compared to epirubicin-treated group.
Wen-Tsan Chang contributed to conception and design,
analysis and interpretation, data collection, and writing of the
paper. Hsiao-Ling Cheng and Bau-Shan Hsieh contributed to
analysis and interpretation and data collection. Chien-Chih
Chiu contributed to analysis and interpretation of data and
critical revision of the paper. King-Teh Lee and Kee-Lung
Chang contributed to conception and design, analysis and
interpretation, critical revision of the paper, and obtaining
funding.
Acknowledgments
This work was supported by National Science Council of Taiwan Grants NSC98-2314-B-037-028-MY1 and NSC98-2314B-037-028-MY2, the Kaohsiung Medical University Grant
KMU-Q101025, and the Kaohsiung Medical University Hospital Grant KMUH-96-6R33.
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The cotreatment also caused a decrease in autophagy, but
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However, clinical results of systemic single-epirubicin
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Therefore, the present study shows the coadministration
of epirubicin and progesterone might be a feasible and
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