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Introduction
Pancreatic cancer is a highly-malignant neoplasm
characterized by locally-advanced, unresectable disease or
metastasis at the time of diagnosis. Over the last three decades,
the 5-year survival of patients with pancreatic cancer has
been <5% without significant
improvement[1]. This poor prognosis is attributed to the high incidence of metastatic
disease at diagnosis. Consequently, major improvement in the
outlook of this disease will depend on the development of
more effective drug therapies. Systemic therapy with
gemcitabine (2´,2´-difluorodeoxycytidine), a deoxycytidine
analog used for pancreatic cancer, has not increased the
median survival of patients beyond 6 months, and often leads
to resistance[2]. Therefore, an effective treatment modality
for this devastating disease is urgently needed.
The potent antitumor activity of enediyne antibiotics has
been the focus of attention because of their unique ability to
damage the DNA of tumor cells by inducing single-strand
and/or double-strand breaks through free radical attacks on the
deoxyribose moieties in DNA[3]. Lidamycin (LDM; also named
C-1027) is a member of the enediyne antibiotic family, which was
produced by a Streptomyces globisporus C-1027 strain isolated
in China[4,5]. The LDM molecule contains an enediyne
chromophore responsible for the extremely potent bioactivity and a
non-covalently-bound apoprotein, which forms a hydrophobic
pocket for protecting the
chromophore[6,7]. LDM shows extremely potent cytotoxicity, anti-angiogenic activity, and marked
growth inhibition of transplantable tumors in
mice[8_11]. It is currently being evaluated in phase II clinical trials as a
potential chemotherapeutic agent in China.
Our previous study showed that LDM was highly active
in targeting the Akt/NF-κB signal pathway and induced
apoptosis and cell cycle arrest in human pancreatic cancer
cells. Moreover, LDM could suppress the growth of xenografts
in athymic nude mice[12]. In the present study, we found that
LDM could potentiate the growth inhibition induced by
gemcitabine in human pancreatic cancer cells, and that the
synergy might be associated with NF-κB downregulation.
Materials and methods
Cells, reagents, and drugs Human pancreatic cancer
cell lines PANC-1 and SW1990 were maintained in culture
with Dulbecco's modified Eagle's medium (DMEM; Gibco
BRL, Grand Island, NY, USA) supplemented with 10%
heat-inactivated fetal bovine serum (Sigma, St Louis, MO, USA),
100 U/mL penicillin, and 100 μg/mL streptomycin at 37
°C in a humidified atmosphere containing 5%
CO2. LDM was provided by Professor Lian-fang JIN from the Institute of
Medicinal Biotechnology (Chinese Academy of Medical
Sciences, Beijing, China). Gemcitabine was the product of
Lilly France SA (Lille, France).
Cell proliferation assay The PANC-1 and SW1990 cells
were plated in triplicate in a 96-well plate with 3000 cells/well
and 4000 cells/well, respectively. After overnight incubation,
the triplicate wells were treated with gemcitabine, LDM, and
a combination of both for 48 h. The effects on cell growth were
examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay. In brief, 20 μL MTT solution (5
mg/mL in phosphate-buffered saline [PBS]; Sigma, USA) were added
to each well and incubated for 4 h at 37
°C. The MTT formazan was dissolved in 150
μL DMSO and absorbance was measured by a microplate reader (Multiskan MK3,
Thermo Labsystem, USA) at a wavelength of 570 nm.
In order to calculate the coefficient of drug interaction
(CDI), the following equation was used:
CDI=AB/A×B,
where AB is the cell survival ratio of the combination
group, A is the cell survival ratio of the LDM group, and B is
the cell survival ratio of the gemcitabine group. CDI <1
indicates the synergistic effect of the drugs.
Analysis of apoptosis by terminal uridine
deoxynucleotidyl transferase dUTP nick end labeling
The nuclear DNA fragmentation of apoptotic cells was measured by TUNEL assay
(DeadEnd colorimetric TUNEL system, Promega Madison,
USA). Briefly, the cells were harvested, washed in PBS,
resuspended in PBS, and added to the poly-lysine-coated
slides. The cells were then fixed in 4% paraformaldehyde,
permeabilized in 0.2% Triton X-100, and incubated with
terminal deoxynucleotidyl transferase incubation buffer for 60
min in a 37 °C humidified chamber for 3´-OH labeling. The
cells that bound with streptavidin horseradish peroxidase
(HRP) and were stained with
3,3´-diaminobenzidine-tetrachloride were analyzed using a fluorescence microscope.
Fluorescein-isothiocyanate-Annexin V/propidium iodide
apoptosis assay The cells were harvested and resuspended
in 200 μL binding buffer. Then 10 μL
fluorescein-isothiocyanate (FITC)-labeled, enhanced Annexin V (Baosai Biotechnology,
Beijing, China) and 100 ng of propidium iodide (PI) were
added. After incubation in the dark (15 min at room
temperature or 30 min at 4 °C), the samples were diluted with 300
μL binding buffer. Flow cytometry was carried out on a FACScan
instrument (Becton Dickinson, NY, USA), and the data were
processed by WinMDI/PC software.
5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimida-
zolylcarbocyanine iodide staining and confocal microscopy
Following the experimental treatments, the cells seeded on
35 mm dishes were stained with JC-1 (Molecular Probes,
Eugene, Oregon, USA) to determine the state of
mitochondrial membrane potential. Briefly, the culture medium was
removed from the adherent cells, and the monolayers were
rinsed once with DMEM. The cell monolayers were
incubated with DMEM containing 10% serum and 5
μg/mL JC-1 at 37 °C for 30 min. Then the cells were rinsed twice with
DMEM, and images were obtained using a 10× objective on
a confocal microscope (Leica SP2, Leica Microsystems,
Wetzlar, German) excited at 488 nm (for JC-1) set to
simultaneously detect green emissions (510_525 nm) and red
emission (590 nm) channels using a dual band-pass filter.
Caspase-3 activity assay Caspase-3 activity was detected
by the Apo-ONER homogeneous caspase-3/7 assay kit
(Promega, USA). Briefly, the cells were seeded into 96-well
plates (1×104/well). Following treatment with the drugs for
48 h, the cells were washed with ice-cold PBS. A total of 100
μL homogeneous caspase-3 reagent was added to each well.
The contents were gently mixed and incubated for 4 h at
room temperature in the dark. The fluorescence intensity of
the Z-DEVD-R110 substrate was measured at an excitation
wavelength of 498 nm and an emission wavelength of 521
nm using a microplate spectrofluorometer (Polarstar, BMG,
Offenburg, Germany).
Western blotting analysis The total protein extract of
the cells was prepared by incubation for 15 min on ice with
an ice-cold hypotonic buffer containing 50 mmol/L Tris-HCl
(pH7.5), 150 mmol/L NaCl, 2 mmol/L EDTA, 2 mmol/L ethyleneglycol bis(2-aminoethyl ether)tetraacetic acid
(EGTA), 1 mmol/L dithiothreitol, 1% Nonidet P-40, 0.1% SDS,
protease inhibitors (1 mmol/L phenylmethylsulfonyl fluoride,
5 mg/mL aprotinin, 5 mg/mL leupeptin, and 5 mg/mL pepstatin) and phosphatase inhibitors (20 mmol/L
β-glycerophosphate, 50 mmol/L NaF, and 1 mmol/L
Na3VO4). The lysates were centrifuged at 12
000×g for 12 min. Protein samples of an equal amount were denatured with 1 volume
of 6× SDS sample buffer and loaded on SDS-PAGE and
transferred to polyvinylidene difluoride membranes (Millipore,
Bedford, MA, USA). The blots were blocked for 60 min at
room temperature with 5% non-fat milk powder and 0.1%
Tween-20 in PBS, and exposed overnight at 4 °C to a primary
antibody against K-ras, NF-κB, and Bcl-2 (Santa Cruz
Biotechnology, Santa Cruz, CA, USA), respectively. The
blots were washed with TBST (20 mmol/L Tris-HCl [pH 7.4],
150 mmol/L NaCl, and 0.1% Tween-20) for 5 min (3 times) and
exposed for 60 min at room temperature to an appropriate
HRP-linked secondary antibody (Santa Cruz Biotechnology,
USA). Complexes of the primary and secondary antibodies
were visualized using enhanced chemiluminescence
Western blotting detection reagents (Amersham Pharmacia
Biotech, Piscataway, NJ, USA).
RT-PCR Total cellular RNA was extracted from the cells
using TRIzol reagent (Invitrogen, Carlsbad, CA, USA)
following the protocol recommended by the manufacturer.
RT-PCR amplification was performed on the extracted RNA
using the Superscript one-step RT-PCR kit
(Invitrogen, USA) according to manufacturer's instructions. The primer pair
for K-ras was as follows:
5´-ACTTGTGGTAGTTGGAGCTG-3´ (sense) and 5´-CTAACAGTCTGCATGGAGC-3´
(antisense). Reverse transcription was performed at
55 oC for 15 min. The denaturation and amplification conditions were 95 °C for 30
s followed by up to 25 cycles of PCR. Each cycle of PCR
included denaturation at 95 °C for 15 s, annealing at 55
°C for 30 s, and extension at 72
°C for 40 s. After PCR amplification,
the fragments were analyzed by 2% agarose gel
electrophoresis. GAPDH was used as a positive control. The sizes were
estimated by comparison with molecular weight markers.
Statistical analysis The results were expressed as
mean±SD. Treatment effects were compared using Student's
t-test, and differences between means were considered to be
significant when P<0.05.
Results
Gemcitabine-induced growth inhibition was potentiated
by LDM in PANC-1 and SW1990 cells The growth of the
PANC-1 and SW1990 cells treated with LDM (1 nmol/L),
gemcitabine (500 nmol/L), or a combination of both was
determined by MTT assay. The dose used in the present study
was chosen based upon a preliminary dose escalation study.
A significant reduction in growth was observed in both cells
treated in combination compared with treatment with LDM
or gemcitabine alone (Figure 1). The CDI was less than 0.7,
which means that the 2 drugs have a synergistic effect.
Gemcitabine-induced apoptosis was sensitized by LDM
in PANC-1 and SW1990 cells We observed the induction
of apoptosis in the pancreatic cancer cells treated with either
gemcitabine, LDM, or a combination of both. Relative to
single agents, the combination treatment induced greater apoptosis in
both cell lines as shown by both the TUNEL analysis and flow
cytometry combined with FITC-Annexin V/PI staining
(Figure 2). The ratios of apoptosis were
59.44%±1.54% and 54.68%±2.62% in the combination groups, whereas those of
the gemcitabine groups were 18.48%±0.94% and
25.79%±2.06% in the PANC-1 and SW1990 cells, respectively. These results are
consistent with cell growth inhibition studies by MTT,
suggesting that the loss of viable cells by LDM and
gemcitabine is partly due to the induction of an apoptotic cell death mechanism.
Gemcitabine-induced apoptosis signaling was
augmented by LDM in PANC-1 and SW1990 cells In an
attempt to explore the mechanism of enhanced apoptotic process
induced by the treatment of cells with LDM and
gemcitabine, we assessed the changes of mitochondrial membrane
potential, caspase-3 activity, and the levels of Bcl-2 in the PANC-1 and
SW1990 cells. Our results showed that the combination
treatment could decrease mitochondrial membrane potential and
enhance caspase-3 activity significantly. The results for the
anti-apoptotic Bcl-2 proteins also showed downregulation
in the combination group relative to the signal-agent
treatment and untreated control (Figure 3).
Effects of gemcitabine, LDM, and a combination on the
K-ras/NF-κB signaling pathway. In the present study, we
found that gemcitabine treatment enhanced NF-κB levels
in PANC-1 and SW1990 cells. However, gemcitabine in
combination with LDM prevented the gemcitabine-induced
NF-κB enhancement through the inhibition of K-ras. Meanwhile, the
levels of K-ras mRNA decreased in both cells after being
treated with a combination of both (Figure 4).
Discussion
Pancreatic cancer is now one of the most common causes
of cancer death worldwide. K-ras mutations are present in up
to 90% of cases of pancreatic
cancer[13]. The expression of mutant K-ras activates the protein kinase B pathway,
resulting in the activation of the NF-κB transcriptional
factor[14]. NF-κB has been shown to inhibit apoptosis in response to
chemotherapeutic agents[15]. Compounds targeting
the NF-κB pathway can sensitize pancreatic tumor cells by counteracting
resistance mechanisms, and therefore, deserve further
evaluation as in the chemotherapy and possible
chemoprevention of pancreatic
cancer[16,17]. In the present study, we found that
gemcitabine in combination with LDM could prevent the
gemcitabine-induced NF-κB enhancement through the
inhibition of K-ras.
Highly-metabolically-active mitochondria were particularly
sensitive and were vulnerable targets to cellular
stress[18]. Membrane depolarization has been widely associated with the
release of the apoptotic factor, cytochrome
c, which amplifies pro-apoptotic caspase cascades, promoting cell
death[18,19]. The anti-apoptotic properties of Bcl-2 and Bcl-xL have been
attributed to their ability to prevent translocation of cytochrome
c to the cytosol, and thus, interfere with the subsequent
activation of cytosolic caspases and
apoptosis[20,21]. In the present study, we found that LDM potentiated the
gemcitabine-induced cell killing by reducing mitochondrial membrane
potential and increasing the caspase-3 activity. The results for
the anti-apoptotic Bcl-2 proteins also showed
downregulation in the combination group relative to the signal-agent
treatment and untreated control.
In conclusion, our current findings have shown a
synergistic effect of gemcitabine and LDM in certain pancreatic
cancer cell lines. The synergy was probably associated with
NF-κB downregulation. Accordingly, further mechanistic
studies would be useful in the treatment of patients with
pancreatic carcinoma.
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