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Introduction
Chronic myelogenous leukemia (CML) is characterized by a reciprocal t (9; 22) chromosomal translocation, known as the
Philadelphia chromosome, which fuses parts of the c-abl gene, located on chromosome 22, and is the hallmark of chronic
myeloid leukemia[1_3]. Today,
P210bcr/abl, the gene product of the resultant bcr/abl hybrid gene, is one of the
most intensively studied signaling proteins in cancer
research[4]. P210bcr/abl-initiated signaling such as
NF-kB decreases the ability of a variety of stimuli to induce apoptosis
in vitro[5,6]. Therefore, it is a new and attractive therapeutic strategy to target
P210bcr/abl and P210bcr/abl signaling.
In recent years, antisense therapy with phosphorothioate oligodeoxynucleotides has emerged as a potentially useful
strategy for inhibiting CML[7]. Bcr/abl phosphorothioate antisense oligonucleotides (PS-ASODN) might be synthesized for
uses as therapeutic agents. The idea is that PS-ASODN would block disease processes by blocking the synthesis of a
protein encoded by bcr/abl. The binding of the PS-ASODN to the bcr/abl mRNA would normally result in the downregulation
of that protein, thus preventing malignant transformation and development of resistance to apoptosis. However, their use as
therapeutic agents is limited by inefficient cellular uptake, scare nuclear internalization, and oligonucleotide
self-aggregation[8].
Curcumin (cur) is a diferuloylmethane derived from the plant
Curcuma longa. It is a potent antioxidant that possesses
both anti-inflammatory and antitumor activities, and
has been regarded as one of the prosperous antitumor
agents[9]. In our previous works, cur has been proved to inhibit the proliferation of K562 cells correlates with the downregulation of the
P210bcr/abl protein[10]. Therefore, we supposed that cur might synergistically inhibit the growth of CML with PS-ASODN. If
there is a combination, what would the action of cur after bcr/abl mRNA be blocked by PS-ASODN?
We selected NF-kB to investigate whether the combination of cur and PS-ASODN exerted a synergistic effect on the
downstream of P210bcr/abl signaling, thus affecting cell growth.
NF-kB, a transcription factor with a critical role in promoting
inflammation and which is connected with multiple aspects of oncogenesis and cancer cell survival, appears to be involved
in the regulation of cell proliferation, control of apoptosis, promotion of angiogenesis, and the stimulation of
invasion/metastasis[5,11]. For further analysis of the inhibitory mechanism of PS-ASODN and cur, we studied the molecular chaperone
heat shock protein 90 (Hsp90), which is important in maintaining the conformation, stability, and function of
P210bcr/abl[12].
In this study, the
P210bcr/abl-positive K562 cell line was
used as a cellular model of CML for drug screening (Figure 1).
Materials and methods
Drugs and antibodies Cur was purchased from Shanghai
No 3 Reagent Company (Shanghai, China;
No 980825). It had a purity of 99%, and was dissolved in DMSO as stock solution.
The sequence of primers are as
follows[13]: bcr/abl anti-sense 5'-GCT GAA GGG CTT TTG AAC TCT GCT-3'; bcr/abl nonsense 5'-CTC TGG ATA GGA TCA
TGT-3'. Both were synthesized by Bioasia Bio-technology Inc (Shanghai,
China); c-Abl, NF-kB, Hsp90, and b-actin antibodies were purchased from Santa
Cruz Biotechnology, Inc (Santa Cruz, CA, USA); Acridine orange (AO) and ethidium bromide (EB) were purchased from
Amresco (Solon, OH, USA), and MTT was from Sigma (St Louis, MO, USA).
Cell culture The human leukemia cell line K562 (purchased from Shanghai Institute of Biochemistry and Cell Biology,
Shanghai, China) was cultured in RPMI-1640 medium, supplemented with 10% fetal bovine serum and gentamicin (80 U/100
mL) in a humidified atmosphere with 5%
CO2 at 37 ºC.
Cell growth inhibition assay
The K562 cells were plated in 0.1 mL in triplicate at a density of
(10_20)×104 cells/mL in 96-well plates and were exposed to the tested agents: PS-ASODN, cur, and a combination of both, which were added at the
selected concentrations to the culture medium with a final volume of 0.2 mL per well for 48 h. Nonsense was used as a control.
At the indicated time, 20 µL of 5 mg/mL MTT solution in
phosphate-buffered saline (PBS pH 7.2) was added to each well for
4 h. After removal of the medium, 170 µL of DMSO was added to each well to dissolve the formazan crystals. The absorbance
at 490 nm was determined using a Biokinetics plate reader (Bio-Rad Laboratories, Hercules, CA, USA). Triplicate wells were
assayed, and standard deviations were determined.
Assessment of apoptosis The K562 cells were plated in 0.1 mL in triplicate at a density of
(10_20)×104 cells/mL in 96-well plates and were exposed to the tested agents: PS-ASODN, cur, or a combination of both. They were added at the selected
concentrations to the culture medium, with a final volume of 0.2 mL per well for 48 h. After the indicated time, 100 cells were
counted under a microscope after AO/EB fluorescent staining to determine the apoptotic
rate[14]. One hundred micrograms of AO and EB were dissolved, respec-tively, in 1 mL PBS (pH 7.2) as stock solution. To further confirm the synergistic
apoptosis, the K562 cells were treated with selected concentrations of 10 µmol/L PS-ASODN, 10 µmol/L cur, or a combination
of both for 48 h. After the indicated time, the apoptotic cells were detected by nuclear morphologic changes using AO/EB
fluorescent staining. Four µL of AO/EB mixture was added with 100 µL of K562 cells on the cover slip, and then the stained
nuclei were viewed under an Olympus microscope (Hatagaya, Tokyo,
Japan) to evaluate the extent of apoptosis (ie cell
shrinkage, nuclear condensation, formation of apoptotic bodies etc).
Protein extraction and Western blotting
After treatment with drugs for 48 h, the cells were collected by centrifugation
and washed 3 times in ice-cold PBS (pH 7.2). The cell
pellets were resuspended in lysis buffer (Tris-HCl 50
mmol/L, pH 8.0, NaCl 150 mmol/L, dithiothreitol 1 mmol/L, EDTA 0.5 mmol/L, NP40 0.1%
(v/v), sodium dodecyl sulfate 0.1%
(w/v) containing protease inhibitors (aprotinin 1 mg/L, leupeptin 2 mg/L, sodium orthovanadate 100 µmol/L and phenylmethyl-sulfonly
fluoride 10%). The protein concentration of each sample was estimated using the Coomassie brilliant blue kit (Nanjing Jiancheng
Biotechnology, Jiangsu, China). After boiling for 5 min, the lysates were subjected to electrophoresis on 8% polyacrylamide
gels and transferred to Hybond-C membranes (Amersham, Arlington Heights, IL, USA) in transfer buffer [Tris 25 mmol/L,
glycine1 90 mmol/L, methanol 20% (v/v)] using a transfer apparatus at 150 mA for 2 h. The membranes were blocked with
blocking-buffer [5% (g/v) non-fat milk] at 4 °C overnight and were incubated with mouse anti-primary antibodies: c-Abl,
Hsp90, NF-kB, and b-actin for 2 h at room temperature. They were washed 4 times in TBST [Tris-buffered saline
supplemented with 0.03% (v/v) Tween-20], and incubated with horse anti-mouse antibody (1:500) for 1.5 h at room temperature; they
were washed 4 times with Tris-HCl (pH 7.2), and the membranes were detected with substrate according to the product kits
(Gene Lab, Singapore). Intensities of the different proteins were quantified by densitometric scanning. All experiments were
repeated at least 3 times and yielded similar results.
Statistical analysis Data were expressed as mean±SD. Jin's formula was used to evaluate the synergistic effects between
the drugs[15]. The formula is:
Q=Ea+b/(Ea+Eb-E
a×Eb); Q is the combination index;
Ea+b which represents the cell proliferative
inhibition rate of the combined drug;
Ea and Eb are signs of the cell proliferative inhibition rate of each drug. After calculation:
Q=0.85-1.15, means indication of simple addiction (+); Q=1.15-2.0
® synergism (+ +); Q>2.0 ® significant synergism (+ + +);
Q=0.85-0.55 ® antagonistic effect (_); Q<0.55
® significant antagonistic effect (_ _). The differences between treatment and
control groups were evaluated with Student's
t-test. The differences were considered significant at
P<0.05.
Results
Effects of cur and/or PS-ASODN on cell growth
Treatment with either cur (5_20 µmol/L) or PS-ASODN (5_20
µmol/L) resulted in an inhibition of cell growth of 40%, 55%, 90%, 30%, 45%, and 65%, respectively (Figure 2A,B). In combina-tion,
the cell number was reduced greatly (Figure 2C), cur (5_20 µmol/L) and PS-ASODN (5_10 µmol/L) showed synergism
(Q>1.15), that is, a combination of 10 µmol/L cur and 10 µmol/L PS-ASODN induced an inhibition of cell growth of 90%, compared
to cur (55%) or PS-ASODN (45%), and showed
synergism (Q=1.19).
Effects of cur and/or PS-ASODN on cell apoptosis
The extent of apoptosis was more significant when the K562 cells were
treated with the combination of cur and PS-ASODN than each treated alone. Exposure of cells for 48 h to cur (5_20 µmol/L)
or PS-ASODN (5_20 µmol/L) induced apoptosis of 25%_55% and 18%_45% (Figure 3A, 3B). However, when exposed to the
combination of cur and PS-ASODN, it significantly increased the percentage of the hallmark of apoptosis, that is, when 20
µmol/L cur (55%) and 20 µmol/L PS-ASODN (45%) were treated in combination, a marked increase (80%) in apoptosis was
induced (Q=1.06), corresponding to a synergistic interaction (Figure 3C). The drug-treated K562 cells were examined for
morphologic evidence of apoptosis using AO/EB fluorescent staining
under an Olympus microscope (×400). Typical apoptotic
features of chromatin condensation and nuclear fragmentation were observed in the treated cells when they were exposed to
both drugs in combination (Figure 4).
Cur and PS-ASODN synergistically downregulated
P210bcr/abl, NF-kB, and Hsp90 expression
To determine whether the potentiality of apoptosis in the K562 cells treated with PS-ASODN combined with cur would be associated with the
downregulation of P210bcr/abl and selected
P210bcr/abl signaling- NF-kB, Western blot was performed to evaluate their expression.
As a result, treatment with a combination of PS-ASODN and cur resulted in a marked reduction of
P210bcr/abl compared to each drug treated alone (Figures 5, 8). When the cells were exposed for 48 h to 10
µmol/L PS-ASODN in combination with increasing concentrations of cur (5_20 µmol/L), the
P210bcr/abl level decreased by 75%, 80%, and 90%,
respectively, compared to the treatment of PS-ASODN (50%) or cur (20%, 40%, and 60%) alone.
The synergistic inhibition was observed in the inhibition of
NF-kB expression (Figures 6, 8), that is, the individual effect
of 10 µmol/L PS-ASODN or (5_20 µmol/L) cur caused a 48%, 31%, 42%, and 48% decrease of
NF-kB expression, respectively. However, when both of the drugs were combin-ed, the
NF-kB level markedly decreased by 66%, 77%, and 86%. This was
consistent with the downregulated proportion of
P210bcr/abl.
Individual treatment of PS-ASODN had little effect on Hsp90
(P>0.05 vs control), whereas (5_20 µmol/L) cur caused a 26%,
72%, and 81% decrease of Hsp90. The Hsp90 treated with cur, or the combination of PS-ASODN and cur, had no significant
difference (P>0.05). This suggested that the downregulation of Hsp90 was through the effect of cur, but not PS-ASODN
(Figures 7, 8).
Discussion
ASODN is the high degree of specificity, without
altering the expression of genes with closely-related sequences. Theoretically, it is one of the ideal therapeutic agents of
CML[9,16]. However, they are very poorly soluble, exhibit low affinity for their target complementary RNA sequences, and
target RNase-H poorly. The solubility of PS-linked ASODN may be improved in that it can direct RNase-H-mediated destrucOur
tion of the target RNA, but this may result in non-antisense inhibition of
growth[13]. What's more, PS-ASODN are very
poorly transported into human cells; liposomes may enhance cellular ASODN uptake, but it seems, can not meet its initial
high expectations. In this study, treatment with PS-ASODN (5_20 µmol/L) alone resulted in an inhibition of cell growth
(30%_60%).
Recently, cur has been commonly studied as a prosperous drug in its anticancer effect. It can inhibit the proliferation and
induce the apoptosis of many kinds of cancer cells
in vitro, and cur is safe, with human clinical trials indicating no
dose-limiting toxicity when administered at doses up to 10
g/d[9,17_19]. In this study, cur synergistically augmented the growth
inhibitory effects of PS-ASODN in human leukemic cells
in vitro. This observed synergistic effect may be clinically important,
and the combinatorial strategies in cancer therapy can provide dramatic improvements in safety and efficacy over monotherapy
regiments.
current results show that the combination of low concentrations of cur (5_20 µmol/L) and PS-ASODN (5_20 µmol/L) were
sufficient to inhibit cell growth by inhibiting proliferation and inducing apoptosis, downregulating
P210bcr/abl, NF-kB, and Hsp90. The consistent downregulation of
NF-kB with P210bcr/abl showed that the effect of the
combination of PS-ASODN and cur also exerted a synergistic inhibition on the downstream signals of
P210bcr/abl, thus affecting the inhibition of cell growth. The possible rationales for combining PS-ASODN and cur were that both drugs inhibit
P210bcr/abl by different mechanisms: PS-ASODN might downregulate the
P210bcr/abl protein by blocking bcr/abl
mRNA, while cur might accelerate the degradation of
Hsp90[12] which is important in maintaining the conformation, stability, and function of
P210bcr/abl, thus downregulating
P210bcr/abl after bcr/abl mRNA was blocked by PS-ASODN.
In summary, PS-ASODN and cur exhibited a synergistic inhibitory effect on the cell growth of K562. The synergistic
growth inhibition was mediated through different mechanisms that involved the inhibition of
P210bcr/abl. The synergistic effect is clinically important and may provide the combinatorial strategies in cancer therapy.
Acknowledgements
We thank Dr Qun-hao ZHANG from Harvard Medical School who helped in our analysis of Western blot, and Dr Dan LU
from Ohio State University for language support.
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