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Introduction
Osteosarcoma is the most common primary malignant
tumor of bone[1]. The current treatment is the combination
of surgery and neoadjuvant chemotherapy using multidrugs,
including methotrexate, doxorubicin, cisplatin and
cyclophosphamide[2]. Although chemotherapy significantly
increases patient survival, there is no effective treatment for
metastatic osteosarcoma. The frequent acquisition of
drug-resistant phenotypes and unwanted side-effects are often
associated with chemotherapy and remain a serious
problem[3]. Therefore, novel therapeutic strategies to increase
chemosensitivity need to be developed.
Interferonα (IFN)-α, which belongs to the type I IFN, is a
multifunctional cytokine exerting immunomodulatory,
antiviral, and anticancer effects[4-6]. It interacts with the
IFN-α/β receptor on the cell surface to induce the activation of
JAK/STAT1 (Janus activated kinase/signal transducers and
activators of transcription 1) pathway to regulate the
transcription of the genes controlling antiproliferative
activities[6].
p53 is a famous tumor suppressor gene which is
commonly destroyed by mutation or deletion in malignant
tumors including osteosarcoma[7]. It controls cell cycle arrest
and apoptosis induced by chemotherapeutic agents
including doxorubicin, by activating Bax, p21, PUMA (p53
Upregulated Modulator of Apoptosis) and Noxa, which are
target genes of p53[8]. Recently, p53 was found to be
correlated with Type I IFN-induced apoptosis in human cancer
cells[9,10]. Thus we hypothesize that
IFNá may cooperate with chemotherapeutic drugs to enhance antitumor effects
by modulating p53-dependent apoptosis. To test this
hypothesis, we utilized the p53-wild and p53-mutant
osteosarcoma cells to examine whether IFNα increases doxorubicin
sensitivity to determine its molecular mechanism. We report
that IFNα enhances doxorubicin sensitivity in osteosarcoma
p53-wild U2OS, but not p53-mutant MG63 cells and define
for the first time a p53-dependent apoptosis as the molecular
mechanism. This work also supports the view that the proper
combination of IFNα and conventional chemotherapeutic
agents may be a rational strategy for improving the
treatment of osteosarcoma with functional p53.
Materials and methods
Cell culture The human osteosarcoma U2OS
cells[11] containing wild p53 and MG63
cells[12] containing mutant p53 were maintained in DMEM (Gibco BRL, Grand Island,
NY, USA) and supplemented with 10% fetal bovine serum,
100 U/mL penicillin, and 100 µg/mL streptomycin at 37
oC in a 5% CO2 humidified atmosphere.
Drugs and reagents Doxorubicin, Hoechst 33258 and
MTT were obtained from Sigma (St Louis, MO, USA).
Human recombinant IFNα2a were purchased from Peprotech
(Rocky Hill, NJ, USA).
MTT assays The logarithmically growing U2OS and
MG63 cells were seeded in 96-well plate at a density of
5×103 cells/well. After overnight growth, the cells were treated
with IFNα and doxorubicin, alone or in combination. After
the indicated time courses, 10 µL of 5 mg/mL MTT was added
into each well followed by incubation for an additional 4 h.
The supernatants were removed and 200 µL DMSO was
added. After the crystals had dissolved, absorbance at 450
nm was measured in the microplate reader.
Flow cytometry analysis The cells were collected, washed
twice with ice-cold phosphate-buffered saline (PBS),
resuspended in cold PBS, and fixed with 70% ethanol. After
fixation overnight and subsequent rehydration in PBS for 30
min at 4 oC, the samples were stained for 30 min in the dark
with 50 µg/mL propidium iodide (Sigma, USA) containing
125 units/mL protease-free RNase, both diluted in PBS in a
flow cytometer (Beckman Coulter, Fullerton, CA, USA).
Morphological analysis of apoptosis The cells were
collected by centrifugation at 1000×g for 5 min, washed twice
with PBS, and stained with 10 µg/mL Hoechst 33258 (Sigma,
USA) for 15 min, followed by examination using a Olympus
fluorescence microscope (Olympus, Shinjuku-ku, Tokyo,
Japan).
DNA fragmentation assay The cells
(3×106) were collected and washed once with PBS. DNA was extracted using
DNAZol reagent (Invitrogen, Carlsbad, CA, USA)
according to the manufacturer's instructions and electrophoresed
on 2% agarose gel containing 0.5 µg/mL ethidium bromide.
The gel was photographed with UV illumination.
Western blot analysis The cells were collected and
lysed with the lysis buffer [10 mmol/L Tris-HCl (pH 7.4), 5
mmol/L MgCl2, 1 mmol/L EDTA, 25 mmol/L NaF, fresh 100
µmol/L Na3VO4 and l mmol/L dithiothreitol]. An equal amount
of protein determined by Bradford assay was resolved by
SDS-PAGE and transferred onto polyvinylidene difluoride
(PVDF) membranes (Roche, Grenzacherstrasse, Basel,
Switzerland). The blots were incubated with primary
antibody overnight at 4 oC, followed with 3 washes in TBST [20
mmol/L Tris-HCl (pH 7.6), 150 mmol/L NaCl, 0.1% Tween-20]
for 5 min, and then incubated with horseradish
peroxidase-conjugated secondary antibody for 1 h at room temperature.
Signals were detected using enhanced chemiluminescence
detection system (Amersham Pharmacia Biotech,
Piscata-way, NJ, USA) and exposed to X-ray film (Kodak, Shanghai,
China). All the antibodies were obtained from Santa Cruz
Biotechnology (Santa Cruz, CA, USA).
RT-PCR Total RNA was extracted with TRIZol reagent
(Invitrogen, Carlsbad, CA, USA) according to the
manufac-turer's recommendations. For the RT reaction, 2 µg RNA
was combined with 0.5 µg oligo
(dT)15 (15 µL total volume). The mixture was incubated at 70
oC for 5 min and placed on ice. Then 5 µL 5× M-MLV (moloney murine leukemia virus)
reaction buffer, 1.25 µL 4× dNTP (10 mmol/L ), 1 µL M-MLV
(Promega, Madison, WI, USA; 200 U/µL), 0.625 µL
RNaseOUT (40 U/µL ) was added (25 µL total volume). The
tube was incubated at 42 oC for 60 min and then at 75
oC for 10 min for termination. The PCR
reaction was performed in the presence of
Taq DNA polymerase, dNTP mix, and PCR buffer
primers (all from Invitrogen, USA). After denaturation at
94 oC for 2 min, the samples underwent 30 cycles of
amplification (1 min at 94 oC, 1 min at 58
oC for p53 or 55 oC for b-actin, and 1 min at 72
oC) with a 10 min extension at 72
oC following the last cycle. The sense and antisense primers
were: 5'-CAG CCA AGT CTG TGA CTT GCA CGT AC-3' and
5'-CTA TGT CGA AAA GTG TTT CTG TCA TC-3' for p53 and
5'-ACT ACC TCA TGA AGA TCC TC-3' and 5'-CTA AAG ATT GCG TGG CGA GG-3' for
b-actin. Products were electrophoresed on 1.5% agarose gels containing 0.5 µg/mL
ethidium bromide.
Small interfering RNA transfection The U2OS cells
were plated onto 6-well plates at a density of
3×105 cells/well with growth medium without antibiotics. After overnight
incubation, transfection was performed at a confluency of
50% by using Opti-MEM media (Invitrogen, Carlsbad, CA,
USA), Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA),
and specific or non-specific siRNA for p53 according to the
manufacturer's recommendations. Six hours later, the
medium was replaced with growth medium without antibiotics.
After transfection for 20 h, the cells were trypsinized and
sub-seeded onto 96 well plates. After incubation for another
4 h, the cells were treated as described for the MTT assay.
The p53 siRNA duplexes were synthesized by Genepharma
(Shanghai, China). The mRNA sequence to be targeted by
p53-siRNA was 5'-CTA CTT CCT GAA AAC AAC G-3'.
Results
IFNα enhances doxorubicin-induced cytotoxicity in
p53-wild U2OS, but not p53-mutant MG63 cells We first
determined whether IFNα could enhance
doxorubicin-induced cytotoxicity using MTT assay. After treatments with
IFNα, doxorubicin or both for 72 h, the proliferation of the
U2OS cells was not found to be inhibited by IFNα and was
slightly suppressed by doxorubicin. However,
doxorubicin-induced cytotoxicity was significantly enhanced by
IFNα (Figure 1). In contrast, such effects were not observed in
p53-mutant osteosarcoma MG63 cells (Figure 1). The MTT
results show that IFNα enhances doxorubicin-induced
cytotoxicity in p53-wild U2OS, but not p53-mutant MG63 cells.
The results indicate that p53 may contribute to this
phenomenon.
IFNα enhances doxorubicin-induced apoptosis in
p53-wild U2OS, but not p53-mutant MG63
cells We next used flow cytometry analysis to evaluate the effect of
IFNα on doxorubicin-induced apoptosis in U2OS and MG63 cells. The
fraction of sub-G1 cells is a dependable way of determining
cells undergoing apoptosis[13]. IFNα did not induce
obvious apoptosis, but notably increased doxorubicin-induced
apoptosis in p53-wild U2OS cells (Figure 2A), but not
p53-mutant MG63 cells (Figure 2B). Moreover, Hoechst33258
staining, which stains nuclei to manifest apoptotic
morphological change, revealed a higher level of nuclear
condensation and fragmentation in the U2OS cells treated for 72 h with
the IFNα/doxorubicin combination than either alone (Figure
3A). We next used agarose gel electrophoresis to further
confirm apoptosis. The internucleosomal DNA
fragmentation was observed in the U2OS cells treated with the
IFNα/doxorubicin combination for 72 h, compared with either alone
(Figure 3B). In contrast, such results were not obtained for
the p53-mutant MG63 cells (data not shown). These results
from different methods strongly indicated that
IFNα enhanced doxorubicin-induced apoptosis in p53-wild U2OS, but not
p53-mutant MG63 cells.
Caspase-3 is a key executor of apoptotic cell death
signals by selectively cleaving proteins, such as poly
(ADP-ribose) polymerase (PARP). Both caspase-3 activation and
PARP cleavage are hallmarks of apoptosis. We next used
Western blotting for their detection them. Caspase-3 and
PARP were cleaved to yield 17 and 85 kDa fragments in
response to the IFNα/doxorubicin combination in the U2OS
cells, respectively (Figure 3C).
IFNα activates p53 as a transcription factor in
response to doxorubicin in osteosarcoma U2OS cells
To determine whether IFNα affects the doxorubicin-induced activation of
the p53 pathway, Western blotting was applied to examine
the expression of p53 and well-known transcriptional target
genes of p53, such as Bax, Bcl-2, Mdm2, and p21. The p53
protein level was unaltered by IFNα, but was enhanced by
doxorubicin and further augmented by the
IFNα/doxorubicin combination. The expression of pro-apoptotic Bax and
p21 was further increased by the IFNα/doxorubicin
combination than either alone. The Mdm2 expression level was
also increased. Adversely, the expression of anti-apoptotic
Bcl-2 was decreased by the IFNα/doxorubicin combination
compared with either alone (Figure 4A). In contrast, such
results were not observed in the MG63 cells (Figure 4A). In
addition, RT-PCR showed that the p53 mRNA level was
increased greatly by doxorubicin, but was not further
augmented by the IFNα/doxorubicin combination in the U2OS
cells (Figure 4B), suggesting that the combination-induced
p53 protein upregulation was mediated in a
post-transcriptional manner.
p53 silencing mediated by small interfering RNA in
U2OS cells results in the decrease of cytotoxicity and
apoptosis induced by the IFNα/doxorubicin
combination To study the presence of the
IFNα/b receptor in U2OS and MG63 cells, Western blotting was used to detect its expression.
The IFNα/b receptor was found to be expressed equally in
both cell lines (Figure 5A). To examine whether p53 was
required for this enhanced apoptosis, we next used
p53-specific siRNA transfection to suppress p53 expression in the
U2OS cells. p53 siRNA effectively inhibited p53 protein
expression in the U2OS cells treated with the
IFNα/doxorubicin combination (Figure 5B). This p53 silencing significantly
reduced the combination-induced cell death (Figure 5C).
Furthermore, as key markers of apoptosis, caspase-3
activation and PARP cleavage were also completely suppressed in
U2OS cells where p53 expression had been suppressed,
compared with the nonspecific control (Figue 5D). These results
show that the enhanced apoptosis induced by the
IFNα/doxorubicin combination was p53-dependent.
Discussion
IFNα is an approved treatment option for tumor therapy,
however; biological activity remains elusive. For example,
IFNα induces cell cycle arrest, triggers apoptosis, and
increases chemotherapy-induced cytotoxicity in specific
cancer cells[14-16]. Recently, IFNα was reported to directly
suppress the growth of some osteosarcoma
cells[17]. However, the role of IFNα in the chemosensitivity of human
osteosarcoma is largely unknown. In the present work, we showed
that IFNα alone unaltered cell growth but promoted
doxorubicin-induced cytotoxicity pronouncedly in osteosarcoma
U2OS cells containing wild p53. Furthermore, the enhanced
cytotoxicity was demonstrated to be mediated by apoptosis
using FACS, Hoechst 33258 staining, DNA fragmentation,
caspase-3 activation and PARP cleavage. In contrast, such
effects were not observed in the p53-mutant MG63 cells.
Although IFNα can directly induce apoptosis in a large group
of tumor cells, such an effect was not observed in the U2OS
and MG63 cells. This is not unusual because these 2 cell
lines are both null of ARF (Alternative Reading Frame)
[18], which is required for IFNα-induced apoptosis in specific cell
types[19].
Although neoadjuvant chemotherapy shows a
promising efficacy in treating osteosarcoma, the resistance against
chemotherapy and drug-induced side-effects remain serious
problems[2]. The topoisomerase II inhibitor doxorubicin is
an antitumor drug widely used in treating human
osteosarcoma[1]. Topoisomerase II is a nuclear enzyme that functions
during both DNA replication and
transcription[2]. Doxorubicin is able to induce DNA damage and leads to cell cycle
arrest or apoptosis by activating
p53[2]. As a tumor suppressor gene which is often disrupted in human malignancies
including osteosarcoma[8,20], the p53 gene product is also
involved in type I IFN-induced
apoptosis[9,10]. Based on these findings and our result that the enhanced apoptosis
occurred in the p53-wild U2OS, but not p53-mutant cells, we
hypothesize that p53 may contribute to the
IFNα/doxorubicin combination-induced apoptosis. Our results showed that
the enhanced apoptosis in response to IFNα/doxorubicin
combination in the U2OS cells was associated with an
accumulation of the p53 protein, which confirmed our hypothesis.
The Mdm2-p53 feedback loop is the main mechanism in
the regulation of the p53 level[21]. Mdm2, another
transcriptional target of p53, inhibits p53 by directly binding to it to
antagonize its activity and enhance its degradation.
Adversely, when the nuclear p53 level is elevated, it
activates the transcription of the Mdm2
gene[21]. After treatment with the combination, Mdm2 expression was
consistent with p53 upregulation. This is possibly because p53
upregulation induced Mdm2 expression according to this
negative feedback loop. However, the p53 mRNA level did
not further increase following the IFNα/doxorubicin
combina-tion, suggesting that p53 upregulation is mediated in a
post-transcriptional manner. However, the exact mechanism is
not clear.
Bax and Bcl-2, members of the Bcl-2 family, exert
pro-apoptotic or anti-apoptotic functions respectively to
regulate p53-dependent apoptosis[22]. The Bcl-2 protein is able
to repress a number of apoptotic death
programs[23]. The 21 kDa protein partner Bax, which overexpresses to counter the
death repressor activity of Bcl-2, can enhance apoptosis.
The ratio of Bcl-2 to Bax determines survival or death
following an apoptotic stimulus[23-24] In this study, the
combination subsequently increased Bax and decreased Bcl-2
expres-sion resulting from p53 upregulation. The wild-type p53
gene is a negative regulator of cell growth by the
transcriptional activation of p21 which plays a crucial role in
controlling DNA repair, cell differentiation, and apoptosis in
response to p53 activation[25]. Furthermore, the expression
of p21 was also further increased by the combination. These
events may induce the mitochondrial permeability transition,
which can release cytochrome c and culminate in apoptotic
cell death[22].
Caspase-3 is a key executor of the apoptotic
machinery[26]. Once activated by apoptotic signals, caspase-3 is
proteolytically cleaved to active its substrates, such as PARP,
resulting in the activation of the DNA fragmentation of
apoptosis[27]. Our study showed that
IFNα markedly promoted caspase-3 activation and PARP cleavage in
doxorubicin-treated U2OS cells, suggesting that a caspase-3
activation pathway was involved.
Although we proved that p53 activation is involved in
enhanced apoptosis induced by the IFNα/doxorubicin
combination, whether it is required for this effect remains
unclear. We show here that siRNA-mediated p53
knockdown markedly decreased the apoptotic response to the
IFNα/doxorubicin combination, as determined by the
decrease of caspase-3 activation and PARP cleavage,
indicating that p53 function is required for the
IFNα/doxorubicin combination-induced cytotoxicity and apoptosis.
In conclusion, we demonstrated that IFNα enhanced the
sensitivity of human osteosarcoma U2OS cells to
doxorubicin by apoptosis, and defined a p53-dependent pathway as
the underlying mechanism. The combination of IFNα and
standard chemotherapeutic agents may help to achieve
enhanced chemosensitivity and reduce the side-effects in
treating osteosarcoma with functional p53.
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