Combined antiproliferative activity of imatinib mesylate (STI-571) with radiation or cisplatin in vitro
Little is known about the interaction of novel anticancer drugs with other treatment modalities. The aim of this study was to examine the effect of combining imatinib mesylate (STI-571) with radiation or cisplatin on the survival of two human solid tumor cell lines – SKNMC cells derived from Ewing s...
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irk-123456789-1385772020-10-17T00:52:45Z Combined antiproliferative activity of imatinib mesylate (STI-571) with radiation or cisplatin in vitro Yerushalmi, R. Nordenberg, J. Beery, E. Uziel, O. Lahav, M. Luria, D. Fenig, E. Original contributions Little is known about the interaction of novel anticancer drugs with other treatment modalities. The aim of this study was to examine the effect of combining imatinib mesylate (STI-571) with radiation or cisplatin on the survival of two human solid tumor cell lines – SKNMC cells derived from Ewing sarcoma and breast cancer MCF-7 cells. Methods: Cell proliferation was determined using the sulphorodamine B cytotoxicity assay. Cell cycle analysis was performed with flow cytometry. Apoptosis was determined using a commercial cell death ELISA plus kit. Phosphorylated AKT, which has been suggested to be involved in radiation resistance, was detected by Western blot analysis. Results: Exposure of SKNMC cells to STI-571 resulted in a dose-dependent antiproliferative effect and a decrease in phosphorylated AKT expression. There was no evidence of apoptosis. The combination of STI-571 with radiation or cisplatin had an additive antiproliferative effect in SKNMC cells (60% reduction in cell number). A similar effect was observed in human MCF-7 breast cancer cells. Conclusion: STI-571 improves the outcome of cisplatin or irradiation treatment in vitro. AKT pathway may play a role in the additive effect of STI-571 and irradiation. Цель: оценить антипролиферативный эффект иматиниба (STI-571) в комбинации с облучением или цисплатиной по отношению к двум клеточным линиям – клеткам линии SKNMC, полученным из саркомы Эвинга, и клеткам рака молочной железы человека линии MCF-7. Методы: для оценки пролиферации клеток применяли метод анализа цитотоксичности с использованием сульфородамина B. Для анализа распределения клеток по фазам клеточного цикла применяли метод проточной цитометрии, апоптоза – с применением коммерческого набора для проведения ИФА. Уровень фосфорилированной киназы АКТ, предположительно связанной с радиорезистентностью, определяли методом Вестерн-блот анализа. Результаты: инкубация клеток SKNMC STI-571 приводила к дозозависимому антипролиферативному эффекту и снижению фосфорилирования AKT, но не апоптозу клеток. Комбинированное применение STI-571 и обления или цисплатины оказывало дополнительное антипролиферативное воздействие на клетки линии SKNMC (60% уменьшения количества клеток). Аналогичные эффекты отмечали на клетках линии MCF-7. Выводы: обработка опухолевых клеток STI-571 усиливает эффект обления и цисплатины in, причем таковой может быть опосредован сигнальным каскадом AKT 2007 Article Combined antiproliferative activity of imatinib mesylate (STI-571) with radiation or cisplatin in vitro / R. Yerushalmi, J. Nordenberg, E. Beery, O. Uziel, M. Lahav, D. Luria, E. Fenig // Experimental Oncology. — 2007. — Т. 29, № 2. — С. 126–131. — Бібліогр.: 29 назв. — англ. 1812-9269 http://dspace.nbuv.gov.ua/handle/123456789/138577 en Experimental Oncology Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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Original contributions Original contributions |
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Original contributions Original contributions Yerushalmi, R. Nordenberg, J. Beery, E. Uziel, O. Lahav, M. Luria, D. Fenig, E. Combined antiproliferative activity of imatinib mesylate (STI-571) with radiation or cisplatin in vitro Experimental Oncology |
| description |
Little is known about the interaction of novel anticancer drugs with other treatment modalities. The aim of this study was to examine the effect of combining imatinib mesylate (STI-571) with radiation or cisplatin on the survival of two human solid tumor cell lines – SKNMC cells derived from Ewing sarcoma and breast cancer MCF-7 cells. Methods: Cell proliferation was determined using the sulphorodamine B cytotoxicity assay. Cell cycle analysis was performed with flow cytometry. Apoptosis was determined using a commercial cell death ELISA plus kit. Phosphorylated AKT, which has been suggested to be involved in radiation resistance, was detected by Western blot analysis. Results: Exposure of SKNMC cells to STI-571 resulted in a dose-dependent antiproliferative effect and a decrease in phosphorylated AKT expression. There was no evidence of apoptosis. The combination of STI-571 with radiation or cisplatin had an additive antiproliferative effect in SKNMC cells (60% reduction in cell number). A similar effect was observed in human MCF-7 breast cancer cells. Conclusion: STI-571 improves the outcome of cisplatin or irradiation treatment in vitro. AKT pathway may play a role in the additive effect of STI-571 and irradiation. |
| format |
Article |
| author |
Yerushalmi, R. Nordenberg, J. Beery, E. Uziel, O. Lahav, M. Luria, D. Fenig, E. |
| author_facet |
Yerushalmi, R. Nordenberg, J. Beery, E. Uziel, O. Lahav, M. Luria, D. Fenig, E. |
| author_sort |
Yerushalmi, R. |
| title |
Combined antiproliferative activity of imatinib mesylate (STI-571) with radiation or cisplatin in vitro |
| title_short |
Combined antiproliferative activity of imatinib mesylate (STI-571) with radiation or cisplatin in vitro |
| title_full |
Combined antiproliferative activity of imatinib mesylate (STI-571) with radiation or cisplatin in vitro |
| title_fullStr |
Combined antiproliferative activity of imatinib mesylate (STI-571) with radiation or cisplatin in vitro |
| title_full_unstemmed |
Combined antiproliferative activity of imatinib mesylate (STI-571) with radiation or cisplatin in vitro |
| title_sort |
combined antiproliferative activity of imatinib mesylate (sti-571) with radiation or cisplatin in vitro |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| publishDate |
2007 |
| topic_facet |
Original contributions |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/138577 |
| citation_txt |
Combined antiproliferative activity of imatinib mesylate (STI-571) with radiation or cisplatin in vitro / R. Yerushalmi, J. Nordenberg, E. Beery, O. Uziel, M. Lahav, D. Luria, E. Fenig // Experimental Oncology. — 2007. — Т. 29, № 2. — С. 126–131. — Бібліогр.: 29 назв. — англ. |
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Experimental Oncology |
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126 Experimental Oncology 29, 126–131, 2007 (June)
More than 50% of patients with cancer require ra-
diation as adjuvant or palliative treatment. Combining
radiation with cytotoxic chemotherapeutic agents has
become common practice. However, little is known
about the interactions of the novel antitumor drugs,
such as STI-571, with standard treatment [1–4].
STI-571 (imatinib mesylate; Gleevec) has been
found to be effective in the treatment of chronic myeloid
leukemia (CML) and gastrointestinal stromal tumors
[5–9]. In CML, it exerts its antitumor action by inhibiting
the phosphorylation of downstream proteins involved
in BCR-ABL signal transduction. STI-571 also affects
receptor tyrosine kinases, namely c-kit and platelet-
derived growth factor (PDGF) receptors [10].
The effect of the interaction of STI-571 and ra-
diation on Abl-expressing cells remains unclear. One
study found that fibroblasts from Abl knockout mice
have reduced sensitivity to ionizing radiation [11],
whereas later reports contradicted this result [2]. To
explain the possible interaction between radiation and
STI-571, some authors suggested that STI-571 may
be involved in the inhibition of Rad 51 expression. Rad
51 is a component of the DNA repair pathway, and its
reduction would be expected to enhance radiation
sensitivity [12, 13]. However, although STI-571 re-
duced Rad 51 levels in glioma cells, it was unable to
completely eliminate the radiation-induced increase
in Rad 51, suggesting the presence of an additional
signaling process [3].
The activation of the phosphatidylinositol 3 kinase
(PI3K)/AKT signal transduction pathway may be a
major contributor to radioresistance as well as to cis-
platin resistance [14, 15]. AKT, the key protein in this
pathway, was reported to be involved in enhancing
cell proliferation and inhibiting apoptosis [16]. Thus,
the (PI3K)/AKT pathway is a promising target for novel
anticancer agents.
In the present study, we examined the effect of
combined treatment with STI-571 and radiation or
cisplatin on SKNMC, a cell line derived from Ewing
sarcoma, and on the human MCF-7 breast cancer
cell line. SKNMC is characterized by overexpression
of the c-kit receptor [17], whereas MCF-7 cells exhibit
low c-kit-receptor expression. Both cell lines display
radiation sensitivity.
Materials and Methods
STI-571 was kindly donated by Novartis Pharma-
ceutical Inc. (Basel, Switzerland). Sulforhodamine B
(SRB) was obtained from Sigma (St. Louis, MO). RPMI
1640, fetal calf serum (FCS) and antibiotics (penicillin
and streptomycin) were purchased from Biological
Industries (Beth Ha’Emek, Israel). The chemothera-
peutic agent cisplatin (Abiplatin) was obtained from
ABIC (Netanya, Israel).
Cell cultures. The SKNMC cell line, derived from
peripheral primitive neuroectodermal tumor, was
CoMbined antiproliferative aCtivity of iMatinib
Mesylate (sti-571) with radiation or Cisplatin in vitro
R.Yerushalmi1, 2, J. Nordenberg1, 3, E. Beery1, O. Uziel4, M. Lahav1, 4, D. Luria1, E. �enigE. �enig1, 2,*
1Felsenstein Medical Research Center, Rabin Medical Center, Beilinson Campus Petach Tikva, Tel Aviv
University, Sackler Faculty of Medicine, Tel Aviv, Israel
2Institute of Oncology, Rabin Medical Center, Beilinson Campus Petach Tikva, Tel Aviv University,
Sackler Faculty of Medicine, Tel Aviv, Israel
3Endocrinology Laboratory, Rabin Medical Center, Beilinson Campus Petach Tikva, Tel Aviv University,
Sackler Faculty of Medicine, Tel Aviv, Israel
4Department of Internal Medicine A, Rabin Medical Center, Beilinson Campus Petach Tikva, Tel Aviv
University, Sackler Faculty of Medicine, Tel Aviv, Israel
Little is known about the interaction of novel anticancer drugs with other treatment modalities. The aim of this study was to exam-
ine the effect of combining imatinib mesylate (STI-571) with radiation or cisplatin on the survival of two human solid tumor cell
lines – SKNMC cells derived from Ewing sarcoma and breast cancer MCF-7 cells. Methods: Cell proliferation was determined
using the sulphorodamine B cytotoxicity assay. Cell cycle analysis was performed with flow cytometry. Apoptosis was determined
using a commercial cell death ELISA plus kit. Phosphorylated AKT, which has been suggested to be involved in radiation resistance,
was detected by Western blot analysis. Results: Exposure of SKNMC cells to STI-571 resulted in a dose-dependent antiproliferative
effect and a decrease in phosphorylated AKT expression. There was no evidence of apoptosis. The combination of STI-571 with
radiation or cisplatin had an additive antiproliferative effect in SKNMC cells (60% reduction in cell number). A similar effect was
observed in human MCF-7 breast cancer cells. Conclusion: STI-571 improves the outcome of cisplatin or irradiation treatment
in vitro. AKT pathway may play a role in the additive effect of STI-571 and irradiation.
Key Words: STI-571, gleevec, imatinib mesylate, irradiation, cisplatin, cell cycle, phosphorylated AKT, apoptosis.
Received: March 13, 2007.
*Correspondence: Fax: 972-39377311
E-mail: efenig@clalit.org.il
Abbreviations used: CML — chronic myeloid leukemia; PDGF —
platelet-derived growth factor; PI3K — phosphatidylinositol 3 ki-
nase; STI-571 — imatinib mesylate.
Exp Oncol 2007
29, 2, 126–131
Experimental Oncology 29, 126–131, 2007 (June) 12729, 126–131, 2007 (June) 127June) 127) 127 127
kindly donated by Dr. Gad Lavie, Sheba Medical Cen-
ter, Israel. MCF-7, a human breast cancer cell line, was
purchased from ATCC. Both cell lines were cultured in
RPMI 1640 supplemented with 10% FCS and antibiot-
ics (penicillin, streptomycin). The cells were incubated
at 37 °C in a 5% CO2, 95% humidified atmosphere.
Incubation of the SKNMC or MCF-7 cells in the
presence of increasing concentrations of STI-571 (0, 5,
10, 15, 20 µM) for 5 days was performed to determine
the concentration of STI-571 required to cause a 50%
decrease in cell number (IC50).
Cytotoxicity assay. Cells (1.5 x 104/ml) were
seeded in quadruplicate in 24-well plates with increas-
ing concentrations (0, 5, 10 µM) of STI-571 and cul-
tured for 5 days. Twenty four hours after seeding, the
cells were irradiated at increasing doses (0, 200, 400,
600 cGY) with a 6MV linear accelerator (Varian 600C,
Palo Alto, CA, USA). Five days after seeding, cytotoxic-
ity was determined with the SRB assay [18].
To test the combined effect of STI-571 and cispla-
tin, the two agents were added to the culture medium
together for 5 days. STI-571 (5 or 10 uM) was added
to 0.05 and 0.1 µg/ml cisplatin for experiments on the
SKNMC line, and to 0.05 and 0 .25 µg/ml cisplatin for
experiments on the MCF-7 line. Cytotoxicity was tested
on day 5 with the SRB assay.
SrB staining. In brief, the medium was removed,
and cold 10% trichloroacetic acid (TCA) was added for
1 h at 4 °C. The TCA was then removed, and the plates
were rinsed with water and stained with SRB, 4 mg/ml
in 1% acetic acid, as described [12]. The bound SRB
was solubilized in 1 ml of 10 mM unbuffered Tris solu-
tion. Thereafter, 100 µl of each sample were transferred
to a 96-well plate and read at 550 nm with a microtiter
ELISA reader. The results were expressed as percent-
age of the control.
Calculation. The inhibitory effect of each agent
added to the cultures was calculated as follows: In-
hibition (%) = [1-(SRB staining in treated wells/SRB
staining in control wells)] X100.
The theoretical additive inhibitory effect of the
agents a and b was calculcated using the follow-
ing equation: Iab =100 x [1-(1-Ia/100) x (1-Ib/100)]
where Iab is the calculated additive inhibitory effect
expressed as % inhibition. Ia and Ib are the measured
inhibitory effects (%) of each agent acting alone as
compared with that of the control cultures. This equa-
tion was derived assuming the inhibitory agents act
independently on the same target population [19].
Western blot. Phosphorylated AKT was detected
in the cells after induction with FCS as follows. Cells
(1 x 106/dish) were grown in serum-deprived RPMI 1640
for 24 h. STI-571 was then added to the culture for 90 min.
To induce phosphorylation of AKT, FCS was added to the
cell cultures for 1 h. Cells were then harvested, washed
with PBS and lysed with the CHAPS lysis buffer (10 mM
Tris-HCl, pH 7.5, 1 mM MgCl2, 1 mM EGTA, 0.1 mM ben-
zamidine, 5mM β-mercaptoethanol, 0.5% 3-[(3-chol-
amidopropyl)-dimethyl- ammonio]-1-propanesulfonate,
10% glycerol). Protein concentration was determined
using the Bradford assay (Bio-Rad Laboratories, Her-
cules, CA, USA). Equal protein amounts of all samples
(20–40 µg) were resolved on 10% sodium dodecylsulfate
(SDS) and transferred to a polyacrylamide gel and then
to a polyvinylidene difluoride (PVDF) membrane. The AKT
protein or its phosphorylated form was detected with a
specific monoclonal antibody (Cell Signaling Technology,
Beverly, MA) in 1 : 1000 dilution, followed by horserad-
ish peroxidase-conjugated goat-anti-rabbit antibody
(Jackson Laboratories, West Grove, PA, USA). The
SuperSignal®West Pico Chemiluminiscent Substrate kit
(Pierce, IL, USA) was used to visualize the expression of
both proteins, according to the manufacturer’s protocol.
Signals were quantified using Quantity–One software for
Bio-Rad image analysis systems (Bio-Rad Laboratories).
Phosphorylated AKT expression was calculated relative
to the total signal obtained from the AKT protein.
Apoptosis assay. Cells (1.5 x 104/ml) were seeded
in a 24-well plate and treated with STI-571 10 or 15 µM,
cisplatin 0.1 µg/ml, and radiation 400 cGY, alone or
in combination. DNA fragmentation was determined
by nucleosome assessment using a commercial Cell
Death ELISA Plus kit (Roche, Mannheim, Germany),
according to the manufacturer’s instructions. Cell
content was estimated in identical plates using the SRB
method. The data obtained by ELISA were normalized
for cell content; the apoptotic index was calculated as
percentage of the untreated controls.
Statistical analysis. The data are presented as
mean ± SD. Each experiment was performed at least
three times. The data were analyzed with two-way analy-
sis of variance (ANOVA) using SPSS software. A p value of
less than 0.05 was considered statistically significant.
results
Effect of Sti-571 on cell proliferation and
apoptosis. Incubation of the SKNMC or MCF-7 cells in
the presence of increasing concentrations of STI-571
for 5 days resulted in a concentration-dependent de-
crease in cell number.
The concentration of STI-571 required to cause a
50% decrease in cell number (IC50) was about 15 µM
for the SKNMC line and 20 µM for the MCF7 line. As
the dose increased, the sensitivity of the SKNMC
cells to STI-571 increased compared to the sensitivity
of the MCF-7 cells (Fig. 1). Therefore, further evalua-
tion of apoptosis and analysis of the cell cycle were
performed on SKNMC cells only.
The data in the Table show that STI-571 had only a
negligible effect on the apoptotic index.
Effect of Sti-571 on phosphorylated AKt le-
vels. The levels of AKT protein previously reported to
be involved in enhancing cell proliferation, inhibiting
apoptosis, and contributing to radioresistance were
evaluated by Western blot analysis.
As shown in Fig. 2, phosphorylated AKT decreased
dramatically by 72% after 90 min exposure of SKNMC
to 15 µM STI-571, as it has recently been reported by
us, while exploring regulation of telomerase activity
by STI-571 [20].
128 Experimental Oncology 29, 126–131, 2007 (June)
fig. 1. Effect of STI-571 on SKNMC and MCF-7 Cells. SKNMC
and MCF-7 cells (1.5 x 104/ml) were incubated in the presence
of increasing concentrations of STI-571, as described in Mate-
rial and Methods. The surviving fraction of SKNMC and MCF-7
cell lines was determined with the sulforhodamine-B staining
method. Values represent mean ± SD of three independent
experiments, each performed in quadruplicate
fig. 2. Phosphorylated AKT expression in response to STI-571
treatment. Phosphorylated AKT and total AKT protein levels after
SKNMC exposure to 15 uM STI-571 were analyzed by Western
blot, as described in Material and Methods. The figure depicts
one representative experiment (out of 3)
fig. 3. Effect of radiation on SKNMC and MCF-7 Cells. SKNMC
and MCF-7 cells (1.5 x 104/ml) were incubated in the presence
of increasing concentrations of STI-571, as described in Mate-
rial and Methods. The surviving fraction of SKNMC and MCF-7
cell lines was determined by SRB staining. Values represent
mean ± SD of three independent experiments, each performed
in quadruplicate
Exposure of SKnMC and MCF-7 cells to com-
bination of Sti-571 and radiation. Fig. 3 shows
the effect of radiation alone on SKNMC and MCF-7
cancer cells, and Fig. 4 and 5 show the effect of the
combination of STI-571 and radiation. Combined treat-
ment resulted in an additive decrease in cell number,
confirmed by two-way ANOVA. This effect was more
pronounced in the SKNMC than the MCF-7 cells.
Radiation induced a marked increase in the apoptotic
index of SKNMC cells. The addition of STI-571 did not
augment the apoptotic effect of radiation (Table).
Table. Effect of STI-571, cisplatin, radiation and their combination on the
apoptotic index of SKNMC cells*
Modalities alone or combined* Apoptotic index (%)
Control 1
STI-571 10 µM 1.19
STI-571 15 µM 1.28
Cisplatin 0.1 µg/ml 1.27
STI 10 µM cisplatin 0.1 µg/ml 1.78
400 cGY 4.1
STI 10 µM + 400 cGY 3.99
*Cells were treated with STI-571 10 and 15 µM, cisplatin 0.1 µg/ml and ir-
radiation of 400 cGY, alone or in combination. Cells were seeded in a 24-well
plate, and DNA fragmentation was determined by nucleosome assessment
using a commercial Cell Death ELISA Plus kit.
fig. 4. Combined effect of STI-571 and radiation on SKNMC
cell line. Cells (1.5 x 104/ml) were incubated with 5 and 10 uM
STI-571. Twenty-four hours after seeding, the treated and un-
treated cells were irradiated at increasing doses (0, 200, 400,
600 cGY) with a 6MV linear accelerator. The surviving fraction
of SKNMC cell lines was determined by SRB staining. Values
represent mean ± SD of three independent experiments , each
performed in quadruplicate. (a) 5 µM STI-571 and 200 cGY;
(b) 5 µM STI-571 and 400 cGY; (c) 5 µM STI-571 and 600 cGY;
(d) 10 µM STI-571 and 200 cGY; (e) 10 µM STI-571 and 400 cGY;
(f) 10 µM STI-571 and 600 cGY
Experimental Oncology 29, 126–131, 2007 (June) 12929, 126–131, 2007 (June) 129June) 129) 129 129
fig. 5. Combined effect of STI-571 and radiation on MCF-7 cell
line. Cells (1.5 x 104/ml) were incubated with 10 uM STI-571.
Twenty-four hours after seeding, the treated and untreated cells
were irradiated at increasing doses (0, 200, 400, 600 cGY) with a
6MV linear accelerator. The surviving fraction of SKNMC and MCF-
7 cell lines was determined by SRB staining. Values represent
mean ± SD of three independent experiments, each performed in
quadruplicate. (a) 10 µM STI-571 and 200 cGY; (b) 10 µM STI-571
and 400 cGY; (c) 10 µM STI-571 and 600 cGY
Exposure of SKnMC and MCF-7 cells to Sti-571
and cisplatin. Incubation of the SKNMC or MCF-7
cells in the presence of increasing concentrations of
cisplatin resulted in a concentration-dependent de-
crease in cell survival. The concentration of cisplatin
required to inhibit 50% cell growth (IC50) was found
to be around 0.35 µg/ml for the MCF-7 cell line and
0.09 µg/ml for the SKNMC cell line.
Incubation of the SKNMC and MCF-7 cells in the
presence of combined STI-571 and cisplatin for 5 days
yielded an additive cell-killing effect on both cell lines
(Fig. 6, 7), with only a slight increase in apoptosis
compared to each agent alone (Table).
disCussion
To improve the outcome of radiation treatment,
clinicians combine its use with standard cytotoxic
chemotherapeutic agents. Since the introduction of
novel anticancer drugs to the daily treatment arma-
mentarium, researchers have been seeking data on
the potential benefits of their interaction with standard
treatment modalities. The present study shows that
STI-571 and radiation have an additive antiproliferative
effect on SKNMC and MCF-7 human solid tumor cell
lines. The results are in accordance with the report
of Topaly et al. [4] who found a synergistic effect of
STI-571 and radiation in BCR-Abl-positive lymphoid
and myeloid blast crisis cells. However the Abl family
proteins are non receptor tyrosine kinase and different
intracellular mechanisms may be involved.
fig. 6. Combined effect of STI-571 and cisplatin on SKNMC cell
line. STI-571 at concentrations of 5 and 10 uM was added to 0.05
and 0.1 µg/ml cisplatin. (a) 5 uM STI-571 and 0.05 ug/ml cisplatin;
(b) 5 uM STI-571 and 0.1 ug/ml cisplatin; (c) 10 uM STI-571 and
0.05 ug/ml cisplatin; (d) 10 uM STI-571 and 0.1 ug/ml cisplatin.
The surviving fraction was determined by SRB staining. Values
represent mean ± SD of five independent experiments
fig. 7. Combined effect of STI-571 and cisplatin on MCF-7 cell
line. STI-571 at concentrations of 5 and 10 uM was added to
0.05 and 0.25 µg/ml cisplatin. (a) 5 uM STI-571 and 0.05 ug/ml
cisplatin; (b) 5 uM STI-571 and 0.25 ug/ml cisplatin; (c) 10 uM STI-
571 and 0.05 ug/ml cisplatin; (d) 10 uM STI-571 and 0.25 ug/ml
cisplatin. The surviving fraction was determined by SRB staining.
Values represent mean ± SD of five independent experiments
130 Experimental Oncology 29, 126–131, 2007 (June)
Since AKT plays a major role in the PI3 kinase signal
transduction pathway, and also largely contributes to
radioresistance, we sought to determine the effect of
combined treatment on levels of phosphorylated AKT
(the active form of AKT). We noted a marked decrease
in phosphorylated AKT following STI-571 treatment
of SKNMC cells. Our data are in accordance with the
report of Ohashi et al. [21] who found a reduction in
phosphorylated AKT and its downstream targets in
cells expressing mutant platelet-derived growth factor
receptor-alpha.
We speculate that the inhibition of these pathways
may have contributed to the additive antiproliferative
effect of STI-571 and radiation. In light of findings that
p-AKT inhibits apoptosis, we checked apoptosis in the
SKNMC cell line; however, no significant contribution
of the drug to apoptotic death was detected. These
results suggest that STI-571 is cytostatic rather than
cytotoxic to SKNMC cells, and join previous findings of
a possible cytostatic character of STI-571 [22, 23].
The cell response to irradiation is affected by the
cell cycle phase. The mitotic phase is the most sensi-
tive, followed by the G2 phase. Resistance to radiation
gradually increases as the cells proceed through the
late G1 and S phases, reaching a maximum in the late
S phase. In cells with a long G1 phase, a peak of re-
sistance is seen early in G1 [24, 25]. Our earlier study
showed that 4 days of treatment of cells with 15 µM
STI-571 resulted in a fivefold increase in the percent
of cells in G2/M phase (15% vs 3.26%, p = 0.0013).
This increase was accompanied by a concomitant
decrease in cells in the S phase, from 36.5% to 26.9%
(p = 0.0029) [20].
On the basis of these data, we examined the influ-
ence of STI-571 on the cell cycle. Our study revealed
that within 24 h of incubation — the time at which we
irradiated the cells — STI-571 had no influence on the
cell cycle. There was, however, a small but significant
increase in the G2 /M phase 4 days after incubation with
STI-571 [20]. Further evaluation of the effect of cell ir-
radiation 4 days after incubation with STI-571 yielded
no significant improvement in inhibition of cell growth
as compared to radiation 24 h after incubation.
Cisplatin is a major drug in the treatment of malig-
nancy. Our study showed that the addition of a novel
drug to an “old” one augments cell death. Accord-
ingly, it was reported that the combination of STI-571
and cisplatin synergistically inhibited lung [1] or head
and neck [27, 28] cancer cell growth. Regardless of
whether their action is additive or synergistic, STI-571
and cisplatin do not interfere with each other’s antipro-
liferative effect on breast cancer and Ewing’s sarcoma
as was observed in our experiments.
These promising preliminary findings may have im-
portant implications for the treatment of various types
of cancer and should support conducting clinical trials
with this new agent [29]. Further studies are needed
to corroborate the benefit of combining STI-571 with
standard modalities.
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антипролиферативная активность иматиниба (sti-571)sti-571)-571)
в комбинации с облучением или цисплатиной in vitro vitrovitro
Цель: оценить антипролиферативный эффект иматиниба (STI-571) в комбинации �� обл��ением или ци��платиной по отно�е-STI-571) в комбинации �� обл��ением или ци��платиной по отно�е--571) в комбинации �� обл��ением или ци��платиной по отно�е-
нию к дв�м клето�ным линиям – клеткам линии SKNMC, пол��енным и�� ��аркомы �вин�а, и клеткам рака моло�ной �еле��ыSKNMC, пол��енным и�� ��аркомы �вин�а, и клеткам рака моло�ной �еле��ы, пол��енным и�� ��аркомы �вин�а, и клеткам рака моло�ной �еле��ы
�еловека линии MCF-7.MCF-7.-7. Методы: для оценки пролиферации клеток применяли метод анали��а цитоток��и�но��ти �� и��поль��о-
ванием ���льфородамина B. �ля анали��а ра��пределения клеток по фа��ам клето�но�о цикла применяли метод прото�ной цито-B. �ля анали��а ра��пределения клеток по фа��ам клето�но�о цикла применяли метод прото�ной цито-. �ля анали��а ра��пределения клеток по фа��ам клето�но�о цикла применяли метод прото�ной цито-
метрии, апопто��а – �� применением коммер�е��ко�о набора для проведения ИФА. Уровень фо��форилированной кина��ы АКТ,
предполо�ительно ��вя��анной �� радиоре��и��тентно��тью, определяли методом Ве��терн-блот анали��а. Результаты: инк�бация
клеток SKNMC �� STI-571 приводила к до��о��ави��имом� антипролиферативном� эффект� и ��ни�ению фо��форилированияSKNMC �� STI-571 приводила к до��о��ави��имом� антипролиферативном� эффект� и ��ни�ению фо��форилирования �� STI-571 приводила к до��о��ави��имом� антипролиферативном� эффект� и ��ни�ению фо��форилированияSTI-571 приводила к до��о��ави��имом� антипролиферативном� эффект� и ��ни�ению фо��форилирования-571 приводила к до��о��ави��имом� антипролиферативном� эффект� и ��ни�ению фо��форилирования
AKT, но не апопто��� клеток. Комбинированное применение STI-571 и обл��ения или ци��платины ока��ывало дополнительное, но не апопто��� клеток. Комбинированное применение STI-571 и обл��ения или ци��платины ока��ывало дополнительноеSTI-571 и обл��ения или ци��платины ока��ывало дополнительное-571 и обл��ения или ци��платины ока��ывало дополнительное
антипролиферативное во��дей��твие на клетки линии SKNMC (60% �мень�ения коли�е��тва клеток). Анало�и�ные эффектыSKNMC (60% �мень�ения коли�е��тва клеток). Анало�и�ные эффекты (60% �мень�ения коли�е��тва клеток). Анало�и�ные эффекты
отме�али на клетках линии MCF-7.MCF-7.-7. Выводы: обработка оп�холевых клеток STI-571 ���иливает эффект обл��ения и ци��-STI-571 ���иливает эффект обл��ения и ци��--571 ���иливает эффект обл��ения и ци��-
платины in vitro vitrovitro, при�ем таковой мо�ет быть опо��редован ��и�нальным ка��кадом AKT.AKT..
Ключевые слова: STI-571, �ливек, иматиниб, обл��ение, ци��платина, клето�ный цикл, фо��форилированная AKT, апопто��.-571, �ливек, иматиниб, обл��ение, ци��платина, клето�ный цикл, фо��форилированная AKT, апопто��.AKT, апопто��., апопто��.
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