Vol.1, No.4, 320-324 (2009) Health
SciRes Copyright © 2009 Openly accessible at http://www.scirp.org/journal/HEALTH/
Inhibition of proteasome by bortezomib increase
chemosensitivity of bcr/abl positive human k562 chronic
myleoid leukemia cells to imatinib
Baran Yusuf1, Coskun Oztekin2, Bassoy Esen Yonca1
1Department of Molecular Biology and Genetics, Izmir Institute of Technology, Urla, Izmir, Turkey; yusufbaran@iyte.edu.tr
2Department of Family Medicine, Ankara Numune Education and Research Hospital, Çankaya, Ankara, Turkey
Received 18 July 2009; revised 28 July 2009; accepted 14 August 2009.
Chronic myeloid leukae mia (CML ) res ults from a
translocation between chromosomes 9 and 22
which generates the BCR/ABL fusion oncopro-
tein. BCR/ABL has constitutively tyrosine kinase
activity resulting in leukemogenesis. Imatinib, a
competitive inhibitor of the BCR/ABL tyrosine
kinase, is the common treatment of CML. De-
spite the outstanding results of imatinib in the
chronic phase of CML, cases of treatment fail-
ure have been reported, resulting in heteroge-
neous molecular response. Bortezomib is a re-
versible inhibitor of the 26S proteasome induc-
ing cell cycle arrest in G2/M phase, apoptosis by
inhibition of NF-kB. In this study, we examined
the possible synergistic apoptotic effects of the
imatinib/bortezomib combination and the re-
sponsible apoptotic mechanisms induced by
this combination in K562 cells. The results of
this study showed increased cytotoxicity by
XTT assay in combination of imatinib and bor-
tezomib as comp ared to any agent alone. On the
other hand, synergistic apoptotic affects of
combination of these agents were also con-
firmed by changes in caspase-3 enz yme activity
and mitochondrial membrane potential. Taking
together, all the results, confirming each other,
showed that the combination of the imatinib and
bortezomib has considerable synergistic effects
on the apoptosis through increase in caspase-3
enzyme activity and decrease in mitochondrial
membrane potential in human K562 CML cells.
Keywords: Imatinib; Bortezomib; Combination
Therapy; CML, BCR/ABL
Chronic myeloid leukemia (CML) is characterized by a
reciprocal translocation between chromosomes 9 and 22
bringing together BCR and ABL genes to form BCR/
ABL fusion protein. BCR/ABL protein, having constitu-
tive tyrosine kinase activity, is responsible for the
pathogenesis of CML. Imatinib (imatinib mesylate,
STI571, Gleevec) is a tyrosine kinase inhibitor with
good efficacy and recently recommended as first line
therapy for the chronic phase CML. BCR/ABL has a
special ATP binding site close to the substrate proteins
binding region. Imatinib works by binding for blocking
function of BCR/ABL ATP binding site. When the ATP
binding site is filled by imatinib, ATP cannot donate the
phosphate and BCR/ABL can no longer activate down-
stream signaling proteins that promote chronic myeloid
leukemia [1,2,3,4,5]. Although imatinib has an out-
standing outcomes in the chronic phase of CML, cases
of treatment failure has been reported. Resistance to im-
atinib is a major problem in CML patients in blast crisis
phase and has been principally associated with BCR/
ABL tyrosine kinase domain point mutation decreasing
the affinity of imatinib to BCR/ABL protein. In addition,
amplification of the BCR/ABL oncogene, aberrant ce-
ramide metabolism, inhibition of apoptotic pathways,
and reduction in effective intracellular concentrations of
the drug by altered drug efflux or influx are responsible
for imatinib resistance [6,7]. As a result of disease per-
sistence and resistance imatinib monotherapy cannot be
referred to a cure for the majority of patients with CML
[8,9,10,11,12]. The primary and acquired resistance to
imatinib is the developing problem in current CML
treatment and has various underlying mechanisms [1].
On the other hand, the combination of imatinib with
anti-CML agents, may be able to kill drug resistant cells
as highly as drug-sensitive parental BCR/ABL+ leukae-
mic cells, unless the completely refractory to the partner
drugs is developed by leukaemic cells.
The main pathway of protein degradation in eukaryo-
tes is through ubiquitin-proteosome pathway [13,14].
Bortezomib has been common knowledge as a dipeptide
boronic acid, potent and reversible inhibitor of the 26S
proteasome [15,16]. Antitumor activity of bortezomib
B. Yusuf et al. / HEALT H 1 (2009) 320-324
SciRes Copyright © 2009 Openly accessible at http://www.scirp.org/journal/HEALTH/
has been proved in myeloma, myeloid leukemia, lym-
phoma, prostate, breast, colon, and lung cancers. For
patients with myeloma and/or relapsed and refractory
diseases bortezomib shows promising results [17]. Bor-
tezomib induces cell cycle arrest in G2M phase and
apoptosis by inhibition of NF-kB [14,18,19,20]. Studies
strongly implicate the activity of the transcription factor
NF-κB in promoting chemoresistance, cytokine-medi-
ated proliferation, tumor metastasis, and angiogenesis.
Bortezomib, as a proteosome inhibitor, also enhances the
sensitivity of tumor cells to chemotherapy and radiation
and reverses chemoresistance. Induction of apoptosis
involves increased Bcl-2 phosphorylation and cleavage,
accumulation of cyclins A and B1 and increased stability
of p53 and up-regulated p53-induced gene expression
In this study we describe synergistic cytotoxicity
when cells are treated with the imatinib in combination
with bortezomib and responsible signaling pathways that
induce apoptosis in human K562 CML cells.
Cell lines, Growth Conditions, and Drugs. Human
K562 CML cells were obtained from German Collection
of Microorganisms and Cell Cultures. Human K562
CML cells were maintained in RPMI 1640 growth me-
dium containing 10% fetal bovine serum and 1% peni-
cillin-streptomycin at 37 in 5% CO2. Imatinib was a
generous gift from Novartis (USA) and bortezomib was
from Gulhane Medical School, Department of Hematol-
ogy. Imatinib and bortezomib were dissolved in distilled
water and 10 mM stock solutions were prepared.
Measurement of growth by XTT. The IC50 values
of imatinib and bortezomib that inhibited cell growth by
50% were determined from cell proliferation plots ob-
tained by XTT as described previously [7]. Briefly,
2x104 cells were seeded into the 96-well plates that con-
tain 200 µl growth medium and the absence or presence
of increasing concentrations of bortezomib and imatinib,
and incubated at 37˚C in 5% CO2 for 72 hours. After-
wards, they were treated with 40 µl XTT reagent for 4
hours. Then, the absorbances of the samples under 490
nm wavelength were measured by Elisa reader (Thermo
Electron Corporation Multiskan Spectrum, Finland). At
the end, IC50 values were calculated to cell survival
Measurement of caspase-3 activity. Caspase-3 activ-
ity was determined using the caspase-3 colorimetric as-
say (R&D Systems, USA). To start with, the cells that
treated with bortezomib and imatinib for 72 hours were
collected by centrifugation at 1000 rpm for 10 minutes.
Afterwards, pellets were treated with 100 µl of cold lysis
buffer (1X) in order to obtain cell lysate. Then cell lys-
ates were incubated on ice for 10 minutes and they were
centrifuged at 14000 rpm for 1 minute. After that, su-
pernatants were transferred to new eppendorf tubes. For
measuring caspase activity, reaction mixture that include
20 µl assay buffer (5X), 25 µl of sample, 50 µl of steril-
ized water and 5 µl of caspase-3 colorimetric substrate
was prepared in 96-well plates adding and incubated for
2 hours incubation at 37˚C. samples was read under 405
nm wavelength by Elisa reader (Thermo Electron Cor-
poration Multiskan Spectrum, Finland). Absorbances
were normalized to protein levels as determined by
Bradford Assay.
Detection of the loss of mitochondrial membrane
potential (MMP). The loss of mitochondrial membrane
potential was detected by JC-1 mitochondrial membrane
potential (MMP) kit (Cell Technology, USA). The loss
of mitochondrial membrane potential is a hallmark for
apoptosis. Firstly, the cells treated with imatinib and
bortezomib were collected by centrifugation at 1000 rpm
for 10 minutes. Supernatants were discarded and 500 µl
of JC-1 dye was added onto pellets and incubated at
37˚C in 5% CO2 for 15 minutes. Then samples were
centrifuged at 1000 rpm for 5 minutes and 2 ml of assay
buffer was added onto the pellets and they were centri-
fuged for 5 minutes at 1000 rpm. All pellets were resus-
pended with 500 µl assay buffer and 150 µl from each of
them was added into the 96-well plate. The aggregate
red form has absorption/emission maxima of 585/590
nm and the green monomeric form has absorption/ emis-
sion maxima of 510/527 nm. The plate was read in these
wavelengths by fluorescence Elisa reader (Thermo Vari-
oskan Spectrum, Finland).
The cytotoxic effects of imatinib or bortezomib alone
or in combination on K562 cells. IC50 values of imati-
nib and bortezomib were found to be 267 nM (Figure 1)
and 65 nM (Figure 2) in K562 cells exposed to increas-
ing concentrations of imatinib and bortezomib for 72
hours, respectively. There were decreases (from 8 to
82%) in proliferation of K562 cells exposed to increas-
ing nanomolar concentrations of imatinib (from 0,2 nM
to 600 nM) for 72 hours as compared to untreated con-
trols (Figure 1). On the other hand 10-, 50-, and 100 nM
bortezomib resulted in 5-, 36-, and 70% decreases in cell
proliferation (Figure 2), respectively. The same concen-
trations of imatinib with a combination of 65 nM borte-
zomib decreased cellular proliferation significantly
(from 82 to 90%), as compared to untreated controls
(Figure 3a). There were 50% decrease in proliferation of
K562 cells in response to 65 nM bortezomib (Figure
The effects of Imatinib/Bortezomib combination on
caspase-3 enzyme activity in K562 cells. Caspase-3
enzyme activity analyses showed that there were 1.21-
B. Yusuf et al. / HEALT H 1 (2009) 320-324
SciRes Copyright © 2009 Openly accessible at http://www.scirp.org/journal/HEALTH/
and 1.29-fold increase in 0.5- and 5 nM imatinib alone
applied K562 cells, respectively while 65 nM bortezo-
mib increased the caspase-3 enzyme activity 1.24-fold
increase by itself (Figure 4). On the other hand, the
same concentrations of imatinib in combination with 65
nM bortezomib increased the enzyme activity 2.4- and
2.85-fold, respectively (Figure 4).
The effects of combination of Imatinib/Bortezomib
on mitochondrial membrane potential. Treatment with
imatinib/Bortezomib caused a significant loss of MMP,
as measured by increased accumulation of of cytoplas-
mic/mitochondrial form of JC-1, in K562 cells as com-
pared any agent alone. There were 1.19- and 1.31-fold
increase in cytoplasmic/mitochondrial JC-1 in 0.5- and 5
nM imatinib applied K562 cells, respectively, while 65
nM bortezomib decreased MMP 1.16 fold by itself. On
the other hand, the same concentrations of imatinib
Figure 1. Effects of imatinib on the growth of K562 cells.
The IC50 concentration of imatinib was calculated from
cell proliferation plots. The XTT assays were performed
using triplicate samples in at least two independent ex-
periments. Statistical significance was determined using
two-way analysis of variance, and (P < 0,05) was consid-
ered significant.
Figure 2. Effects of bortezomib on the growth of K562
cells. The IC50 concentration of bortezomib was deter-
mined by XTT assay for each cell line as described. The
XTT assays were performed using triplicate samples in at
least two independent experiments. Statistical significance
was determined using two-way analysis of variance, and
(P < 0,05) was considered significant.
with 65 nM bortezomib decreased MMP 1.88- and
2.79-fold, respectively (Figure 5).
The way for treatment of cancer more than one agent is
Figure 3. Effects of bortezomib and imatinib combination
on the growth of K562 cells (a). Effects of 65 nM borte-
zomib by itself on K562 cells as compared to untreated
controls (b). Cytotoxicity was determined by the XTT cell
proliferation test in a 72 hours culture. The XTT assays
were performed using triplicate samples in at least two
independent experiments. Statistical significance was de-
termined using two-way analysis of variance, and (P <
0,05) was considered significant.
Figure 4. Percent Changes in Caspase-3 Enzyme Ac-
tivity in bortezomib and imatinib combination or any
agent alone exposed K562 Cells. The results are the
means of two independent experiments. P < 0,05 was
considered significant.
B. Yusuf et al. / HEALT H 1 (2009) 320-324
SciRes Copyright © 2009 Openly accessible at http://www.scirp.org/journal/HEALTH/
Figure 5. Percent changes in cytoplasmic/mitochondrial
JC-1 in bortezomib and imatinib combination or any
agent alone exposed K562 Cells. The results are the
means of two independent experiments. P < 0,05 was
considered significant.
known as combination therapy. The main aim of the
combination therapy is to get more effective result with
at least two agents than any agent alone. For this purpose,
the agents targeting the different mechanisms and having
effect on different ways are preferred. Several groups
demonstrated that targeting the combination therapy
increases the effect of one agent and it appears a very
good alternative way for the treatment of cancer [22, 23,
24]. Thus the complete destruction of BCR/ABL+ leu-
kemic cells may require BCR/ABL inhibitors in combi-
nation with other therapeutic agents that modify the mo-
lecular pathways that control cell survival. The treatment
of Philadelphia chromosome-positive (Ph+) leukemias
has improved markedly owing to the development of
inhibitors of BCR/ABL tyrosine kinase, as a first mem-
ber tyrosine kinase inhibitors imatinib. However, resis-
tance to imatinib resulting from various molecular me-
chanisms, such as BCR/ABL overexpression, aberrant
ceramide metabolism, mutations within the ima- ti-
nib-binding site of BCR/ABL, expression of a drug ef-
flux pump or compensatory overexpression of related
Src family kinases [4,7].
Bortezomib reduces NF-kb activation and inhibits its
translocation to the nucleus. On the other hand having
the ability to arrest the cells in G2/M, it can increase the
sensitivity of cancer cells to chemotherapy and radio-
therapy. The BCL-2 family plays significant roles as a
key activators of mitochondrial apoptosis in mediating
bortezomib toxicity [14,25].
There were some recent studies showing increased
synergistic apoptotic effects of imatinib and/or borte-
zomib with a combination of other anticancer agents in
hematological malignancies. In isolated chronic lympho-
blastic cells, bortezomib has been shown to induce con-
siderably higher level of apoptosis than either methyl-
prednisolone or fludarabine and the combination of flu-
darabine plus bortezomib increased apoptosis more as
compared to any agent alone, even in fludarabine-resis-
tant cells [15]. In 2008, Wiberg and his coworkers ex-
plored 115 samples of tumour cells that bortezomib is
more active in haematological malignancies than in solid
tumour samples. Their results showed a strong synergy
with arsenic trioxide or irinotecan and bortezomib [13].
In an interesting study by Yong et al., in CD34+ cells of
fourteen CML patients from different phases and in
K562 cells, as a positive control, bortezomib treatment
improved sensitization, upregulated the expression of
TRAIL receptors on quiescent leukemic CD34+ cells and
increased their susceptibility to expanded donor NK
cells [27]. Likewise, another data support a model in
which the combination of sorafenib, multikinase inhibi-
tor, and bortezomib showed synergistic cytotoxicity by
modulating Akt and JNK signaling to activate apoptosis
[28]. On the other hand, bortezomib in combination of
histone deacetylase inhibitor, SAHA, was subjected to
another study demonstrating that bortezomib and SAHA
combination resulted in imatinib-sensitive and -resistant
BCR/ABL positive cells to apoptosis through induction
of JNK and p21CIP1 [29].
To overcome resistance problems in CML, combina-
tions of drugs with imatinib provided an emerging the-
rapeutic concept. The recent study by Cortes and his
coworkers showed effectiveness of imatinib with lon-
afarmib in CML patients who were initially resistant to
imatinib. Combination of imatinib with cytarabine also
showed synergistic cytotoxic effects in CML patients
[30]. It was also shown by our group that imatinib in
combination with docetaxel and fludarabine presented
synergistic apoptotic effects in K562 cells (Unpublished
data). All examinations showed that combination of bor-
tezomib with imatinib induces apoptosis synergistically.
In this study we evaluated the possible synergy of
combination of imatinib and bortezomib. Our data pro-
vide that bortezomib enhanced the chemosensitivity of
BCR/ABL positive human K562 CML cells to imatinib.
Combination therapy can be revealed promising results
for new approaches to underlying mechanism of drug
resistance. The combination of these agents induced
apoptosis through decrease in mitochondrial membrane
potential and increase in caspase-3 enzyme activity.
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