Download The Pro-apoptotic Effects of Thalidomide on a Pancreatic Cancer

C
ANCER
PREVENTION RESEARCH
□ ORIGINAL ARTICLE □
The Pro-apoptotic Effects of Thalidomide on a
Pancreatic Cancer Cell Line
Seong Uk Lim, Dae Eun Kim, Dae Ho Cho, Ju Young Yoon, Ji Hee Lee,
Jun Eul Hwang, Hyun Jeong Shim, Woo Kyun Bae, Sang Hee Cho and Ik-Joo Chung
Department of Hematology-Oncology, Chonnam National University Medical School, Gwangju 501-757, Korea
Thalidomide has been shown to have therapeutic potential in a number of conditions including cancer.
The aim of this study was to evaluate the apoptotic effects of thalidomide on a pancreatic cancer cell
line. Treatment with thalidomide decreased the viability of BxPc-3 cells in dose dependent manner. Using
flow cytometry analysis, determination of whether thalidomide treatment of BxPc-3 cells at concentrations
of 10, 25, and 50 μg/ml affected the number of annexin V-positive cells was evaluated. The percent
of apoptotic cells were 9.5±2.6%, 21.7±3.6% and 29.3±4.7% respectively in the BxPc-3 cells at 10 μg/ml,
25 μ/ml and 50 μg/ml of thalidomide treated for 24 hours. Thalidomide induced the expression of
pro-apoptotic Bax in the BxPc-3 cells and increased the Bax: Bcl-2 ratio. In addition, the results of this
study showed that thalidomide increased apoptosis induced by gemcitabine. (Cancer Prev Res 15, 39-44,
2010)
Key Words: Thalidomide, Apoptosis, Pancreatic cancer
pancreatic cancer carcinogenesis and the related molecular
events, has led to several distinct therapeutic advances, including many novel targeted agents, such as monoclonal antibodies against epidermal growth factor receptor (EGFR), EGFRtyrosine kinase inhibitors, monoclonal antibody against vascular
endothelial growth factor (VEGF), farnesyl transferase inhibitors, and matrix metalloproteinase inhibitors.6∼10)
Thalidomide has been shown to have therapeutic potential
in a number of conditions where there are limited therapeutic
options, including cancer.11,12) The clinical efficacy may be
associated with a number of diverse properties attributed to
thalidomide such as the inhibition of TNF-α synthesis,13)
co-stimulation of T cells,14) and inhibition of angiogenesis.15)
The design and synthesis of thalidomide analogues is an
ongoing research effort to obtain compounds with enhanced
activity/toxicity profiles.16,17) These analogues have been shown
to segregate into at least two distinct classes: the Selective
INTRODUCTION
Pancreatic cancer is a very aggressive disease. While surgery
remains the only potential curative option for this cancer, the
vast majority of patients present with advanced, unresectable
disease. The median survival is up to six months in patients
with advanced disease, and ＜3% of patients are alive at five
years.1) Although it has been demonstrated that gemcitabine
is an effective medication for palliation of symptoms and
prolonging the survival of patients with advanced pancreatic
cancer. Single-agent gemcitabine has shown only a limited
benefit, with 1-year survival rates of 20% and a median overall
survival of around six months.2∼5) Therefore, identification of
a new effective chemotherapeutic regimen for pancreatic cancer
is needed.
The recent elucidation both of the mechanisms involved in
책임저자：정익주, 󰂕 519-809, 전남 화순군 화순읍 일심리 160
화순전남대학교병원 혈액종양내과
Tel: 061-379-7632, Fax: 061-379-7628
E-mail: [email protected]
접수일：2010년 3월 5일, 1차수정일: 2010년 3월 8일,
2차수정일: 2010년 3월 9일, 게재승인일：2010년 3월 12일
Correspondence to：Ik-Joo Chung
Department of Internal Medicine, Chonnam National University Hwasun
Hospital, 160, Ilsim-ri, Hwasun-eup, Hwasun-gun 519-809, Korea
Tel: +82-61-379-7632, Fax: +82-61-379-7628
E-mail: [email protected]
39
40 Cancer Prevention Research Vol. 15, No. 1, 2010
Cytokine Inhibitory Drugs (SelCIDs) consist of 3', 5'-cyclicnucleotide phosphodiesterase (PDE4) inhibitors and the Immunomodulatory Drugs (IMiDs) thought to be mechanistically
similar to thalidomide, act by mechanisms that have not yet
been described. Thalidomide and more recently the IMiD,
CC-5013, have been used successfully to treat patients with
18∼21)
multiple myeloma;
such treatment has been extended to
22∼24)
patients with solid tumors.
Thalidomide analogues are
25∼31)
32)
also being evaluated in vitro,
in vivo and for the
treatment of patients with end-stage cancer. However, there is
little information concerning the direct effect of thalidomide on
solid tumors.
In this study, thalidomide was found to induce apoptosis of
BxPC-3 cells. This activity was associated with alterations in
the balance of pro- and anti-apoptotic bcl-2 family proteins.
MATERlALS AND METHODS
1. Cell culture
The human pancreatic cancer cell line BxPC-3 was obtained
from the Korean Cell Line Bank. The BxPC-3 cells were
cultured in RPMI 1,640 medium (Sigma, St. Louis, MO., USA)
supplemented with 10% fetal bovine serum, 1% penicillin/
streptomycin, and 2 mM L-glutamine. The cultures were
maintained at 37oC in a humidified atmosphere of 95% air and
5% CO2.
2. Thalidomide and other reagents
Thalidomide was obtained from Sigma. A stock solution (20
mg/ml) was prepared fresh in DMSO and diluted directly in
the cultured cells up to 200 μg/ml. The concentration of
DMSO was ＜0.5% in all of the experiments conducted in this
study. The antibodies against Bax, Bcl-2, and Actin were
purchased from Santa Cruz Biotechnology.
3. Cell proliferation assay
All proliferation assays were carried out in triplicate wells
using parallel 96-well microtiter plates. The cells were plated
3
at a concentration of 5×10 cells per well in three parallel
plates with various concentrations of thalidomide for 24 hours.
3
The cells were pulsed with [H]-metylthymidine in each well
during the last 16 hours of culture, harvested, and analyzed
in a liquid scintillation counter (Beckman Instruments Inc.,
Fullerton, CA, USA). The results were expressed as the
mean±SD of results obtained from three different samples, each
performed in triplicate. Data are presented as a percent of the
values obtained from cells cultured under the same conditions
in the presence of DMSO only.
4. Apoptosis assay
Flow cytometry was performed for the apoptosis assay. The
BxPC-3 cells were plated in parallel in 6-well plates at a
concentration of 1×106 cells per plate. After 24 hours, the
adherent and floating cells were harvested by centrifugation.
The cells were treated using the Annexin-V-FITC apoptosis
detection kit (BD Biosciences Pharmingen, San Diego, CA.,
USA) and the apoptotic cells were analyzed by flow cytometry
with FACScan.
5. Western blot analysis
After the appropriate treatments, the cells were harvested by
adding ice-cold cell lysis buffer [1% Nonidet P-40 (NP-40),
50 mM Tris, 150 mM NaCl, 0.25% deoxycholate, 1 mM
EGTA, 1 mM NaVO3, and a protease inhibitor cocktail was
added (Sigma, St. Louis, MO., USA)].
Samples were incubated on ice for 30 min and centrifuged
at 12,000 rpm for 10 min. The Bradford quantification assay
(Bio-Rad, Hercules, CA., USA) was performed on the resulting
supernatant. Equal amounts of protein (30 μg) were loaded
onto a 12% sodium dodecyl sulfate-polyacrylamide electrophoresis gel (SDS-PAGE) and the sample was electrophoresed.
The gel was transferred to nitrocellulose membranes at 100
V for 60 minute. The membranes were blocked by incubation
in 5% skim milk for 1 to 2 hour. Next, the membranes were
incubated with the primary antibodies Bax and Bcl-2 at 4°C
overnight. Secondary antibodies containing the horseradish
peroxidase detection system for chemiluminescence were used
as recommended by the manufacturer.
RESULTS
1. Thalidomide inhibited the proliferation of cultured BxPC-3 cells
To determine the effects of thalidomide on cell growth, the
human pancreatic cancer cell line BxPC-3 was treated with
increasing concentrations of thalidomide or DMSO for 24
hours, and cell survival was assessed by the MTT assay. As
shown in Fig. 1, thalidomide (12.5∼200 μg/ml) treatment
Seong Uk Lim, et al：The Pro-apoptotic Effects of Thalidomide on a Pancreatic Cancer Cell Line
resulted in a concentration dependent inhibition of the proliferation of BxPC-3 cells.
2. Thalidomide induced apoptosis in BxPC-3
cells
Using flow cytometry analysis of thalidomide treatment of
the BxPC-3 cells at concentrations of 12.5, 25 and 50 μg/ml
41
showed the affects of thalidomide on the number of annexin
V-positive cells. Although only a small proportion of the cells
demonstrated spontaneous apoptosis after 24 hour in the
control medium (including DMSO), the percent of apoptotic
cells increased up to 9.5±2.1%, 16.4±3.1% and 22.1±2.9%
in the BxPC-3 cells treated with 12.5 μg/ml, 25 μg/ml and
50 μg/ml of thalidomide, respectively (Fig. 2).
3. Thalidomide induced expression of Proapoptotic Bax in the BxPC-3 cells
Bax and Bcl-2 belong to a multi-gene family of proteins that
play an important role in the regulation of apoptosis. Bcl-2
promotes cell survival, whereas Bax antagonizes this effect.33)
The changes in Bax and Bcl-2 protein expression in BxPC-3
cells, after treatment with thalidomide, were examined. The
protein expression of Bax, not Bcl-2, increased with thalidomide treatment in a dose-dependent manner. The ratio of Bax
to Bcl-2 increased after thalidomide treatment in a dosedependent manner, demonstrating the effects of thalidomide on
Fig. 1. Effects of thalidomide on proliferation of BxPC-3 cells:
Cells were grown in RPMI 1,640 and treated with increasing
concentrations of thalidomide for 24 hours. Data are the
mean±SD of two independent experiments in triplicate. Data
are presented as a percent of the values obtained from cells
cultured under the same conditions in the presence of DMSO
only.
Fig. 2. Thalidomide induced BxPC-3 cell apoptosis: the
BxPc- 3 cells were cultured in medium alone (plus DMSO)
or were incubated with different concentrations of thalidomide
(12.5 μg/ml, 25 μg/ml, 50 μg/ml) for 24 hours and analyzed for annexin V positivity by flow cytometry. Thalidomide
enhanced the annexin V binding in a concentrationdependent manner.
Fig. 3. Effect of thalidomide treatment on protein levels of Bax,
and Bcl-2 determined by immunoblotting. (A) Bar diagram
summarizing the effect of thalidomide on Bax/Bcl-2 ratio. (B)
an equal amount of protein was subjected to Western blotting.
Intensities of the immunoreactive bands were quantified by
densitometric scanning. Data are representative of at least two
independent experiments with similar results.
42 Cancer Prevention Research Vol. 15, No. 1, 2010
Fig. 4. Additive effects of thalidomide on gemcitabine induced
BxPC-3 apoptosis: the cells were cultured in medium alone
(plus DMSO) or were cultured with thalidomide (50 μg/ml),
gemcitabine and thalidomide plus gemcitabine for 24 hour and
analyzed for annexin V positivity by flow cytometry.
apoptosis.
4. Thalidomide increased the apoptosis induced
by gemcitabine
Gemcitiabine is the most widely used drug for pancreatic
cancer. The apoptosis caused by thalidomide, gemcitabine and
combinations of gemcitabine and thalidomide were examined.
As shown in Fig. 4, thalidomide enhanced the apoptosis of
BxPC-3 cells induced by gemcitabine. This result suggests that
thalidomide can be used for the treatment of pancreatic cancer
combined with gemcitabine.
DISCUSSION
Thalidomide (N-phthalyl-glutamic-acid-imide) was first introduced in 1953 as an oral sedative hypnotic.34) After the drug
was withdrawn from the market due to its teratogenicity, the
surprising activity of thalidomide in patients with reactive
lepromatous leprosy stimulated further interest in this drug.35)
Thalidomide has recently been studied for many immune
function disorders as well as cancers. 36∼38,18∼33)
The results of this study showed that thalidomide induced
apoptosis of BxPC-3 pancreatic cancer cells. Thalidomide has
profound effects on the expression of the Bcl-2 family of
proteins. The ratio of Bax: Bcl-2 was significantly increased
with thalidomide treatment. This was mainly due to the
marked increase in Bax protein. The role of Bax in the
39)
induction of tumor apoptosis has been well established. For
example, patients with colorectal carcinoma have reduced
40)
expression of Bax, and its overexpression has been shown to
41)
mediate caspase-dependent apoptosis.
Conversely, the
overexpression of anti-apoptotic proteins, such as Bcl-2, has
been associated with chemoresistance and a poor prognosis in
40,42)
a variety of solid-tumor cell types.
Although it has been demonstrated that gemcitabine is an
effective drug for palliation of symptoms and prolonging
survival in patients with advanced pancreatic cancer, singleagent gemcitabine has shown limited benefit, with objective
response rates of less than 15% and a median overall survival
2∼5)
of around 4.6 months.
Therefore new and more effective
chemotherapeutic regimens are needed for pancreatic cancer.
The results of this study suggest that thalidomide can be used
for the treatment of pancreatic cancer combined with gemcitabine. Thalidomide increased apoptosis induced by gemcitabine. Combination therapy with gemcitabine and thalidomide may provide additional patient benefits for the treatment
of pancreatic cancer.
CONCLUSION
Thalidomide induced the expression of pro-apoptotic Bax in
the pancreatic cancer cells and increased the Bax: Bcl-2 ratio.
In addition, thalidomide increased apoptosis induced by gemcitabine. Therefore, we believe that these data have the potential to provide a novel therapeutic approach for the treatment of pancreatic cancer.
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