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HEMATOPOIESIS
From the Division of Hematology and Medical Oncology,
Tochigi Cancer Center, Tochigi, Japan; Divisions of Molecular
Hematopoiesis and Molecular Biology, Jichi Medical School, Tochigi,
Japan; Division of Molecular Cytogenetics, Department of Clinical
Pathology, Research Institute, International Medical Center of Japan,
Tokyo, Japan; and Division of Chemotherapy, Saitama Cancer Center
Research Institute, Saitama, Japan.
The BCR/ABL tyrosine kinase has been implicated in the pathogenesis
of chronic myelogenous leukemia (CML) and Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia
(ALL). STI571 is a novel anticancer agent that selectively inhibits the
BCR/ABL tyrosine kinase. The cytotoxic effects of STI571 were studied
in combination with antileukemic agents against Ph+
leukemia cell lines, KU812, K-562, TCC-S, and TCC-Y. The cells were
exposed to STI571 and to other agents simultaneously for 5 or 7 days.
Cell growth inhibition was determined by MTT assay. The cytotoxic
effects in combinations at the inhibitory concentration of 80% level
were evaluated by the isobologram. STI571 produced synergistic effects
with recombinant and natural About 90% of chronic myelogenous leukemia (CML),
20% to 30% of adult acute lymphoblastic leukemia (ALL), and 1% to
2% of acute myeloblastic leukemia (AML) show a reciprocal
translocation between chromosome 9 and 22, so-called Philadelphia (Ph)
chromosome.1,2 This translocation results in the
head-to-tail fusion of the breakpoint cluster region (BCR)
gene on chromosome 22 at band q11 with the ABL
proto-oncogene located on chromosome 9 at band q34.3 In Ph+ CML and ALL cases, the BCR/ABL chimeric
transcripts are expressed; in general the P210 BCR/ABL
transcript is expressed in almost all Ph+ CML cases and in
half of Ph+ ALL cases, and the P190 BCR/ABL
transcript is observed in the remaining of Ph+ ALL cases,
although only small amounts of the P190 BCR/ABL
transcript have been reported to be expressed in a majority of
Ph+ CML.4 The BCR/ABL protein has an
antiapoptotic activity and is believed to play a central role in
the development of CML and Ph+ ALL.5-7 The
leukemia-promoting function of this protein requires its deregulated
tyrosine kinase activity, but the precise mechanism of this enzyme to
transform cells is still obscure.5-7
The prognosis of CML and Ph+ ALL is poor.8,9
CML in the chronic phase can be controlled by anticancer agents such as
interferon- With increased understanding of the genetic changes that cause
malignant transformation, new drugs have been developed that might
specifically target the cancer. The BCR/ABL tyrosine kinase could be
such a target for the pharmacologic treatment of CML and
Ph+ ALL, for example, through the use of BCR/ABL tyrosine
kinase inhibitors. Herbimycin A, which effectively reduces
intracellular phosphorylation by BCR/ABL tyrosine kinase,
preferentially inhibits the growth of Ph+ leukemia cell
lines both in vitro and in vivo,11-13 suggesting that
herbimycin A and related compounds may be useful for the treatment of
Ph+ leukemias. However, the selectivity of herbimycin A for
BCR/ABL tyrosine kinase is obscure and clinical application has not yet been performed.
To improve the target specificity of BCR/ABL, many compounds have been
synthesized. STI571 is one of 2-phenylaminopyrimidine derivatives,
which selectively inhibits the tyrosine kinase activity of ABL and
BCR/ABL.14 There was no significant inhibition of the
other numerous protein kinases tested with the exception of the
platelet-derived growth factor receptor14 and the c-Kit tyrosine kinases. STI571 preferentially inhibited the growth of Ph+ leukemia cell lines in vitro.14-16
Injection of Ph+ KU812 and Ph The agents used for the combination are selected on the basis of
single-agent activity, least overlapping toxicity, low
cross-resistance, and additive or synergistic interactions. However,
experimental findings of STI571 in combination with antileukemic agents
are few and controversial.19-22 In the present study, we
investigated the in vitro effects of STI571 in combination with
commonly used antileukemic agents against 4 Ph+ leukemia
cell lines. The dose-response curves for the combinations were obtained
using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay23 and the data at the inhibitory concentration of 80% (IC80) level were analyzed by the isobologram
method (Steel and Peckham).24 The present findings
underline the importance of the design of the combination of STI571
with antileukemic agents for CML and Ph+ ALL.
Cell lines
All 4 Ph+ leukemia cell lines expressed both the P210
BCR/ABL transcript and the P190 BCR/ABL
transcript by reverse transcriptase-polymerase chain reaction (RT-PCR)
using first and nested primer sets.25 The primer sets for
major BCR/ABL transcript detection were: BCR-1S (BCR ex. 13) 5'-ATCCAAGGCTACGGAGAGGC-3', ABL-1AS
(ABL ex.2) 5'-ATGGTACCAGGAGTGTTTCTCC-3' for the first PCR,
and BCR-2S (BCR ex.13) 5'-GGAGCTGCAGATGCTGACCAAC-3' and
ABL-2AS for the nested PCR. The primer sets for minor
BCR/ABL transcripts were: BCR-1mS (BCR ex.1)
5'-CAACAGTCCTTCGACAGC-3' and ABL-1AS for the first PCR, and BCR-2mS
(BCR ex.1) 5'-CAGTGCCATAAGCGCACC-3' and ABL-2AS for the
nested PCR. The thermal cycling profile was 95°C for 12 minutes,
followed by 35 cycles of 95°C for 30 seconds, 62°C for 45 seconds,
and final extension at 72°C for 12 minutes. The nested PCR was
performed for 40 cycles with the same parameters.
Drugs
Inhibition of cell growth by STI571 alone and by the combination of STI571 and the other agents The Ph+ leukemia cells were harvested from the medium and resuspended to a final concentration of 6 × 104 cells/mL of fresh medium containing 10% fetal calf serum (FCS) for TCC-S and KU812, 3 × 104 cells/mL for K562, and 2 × 105 cells/mL for TCC-Y cells. Cell suspensions (100 µL) were dispensed into individual wells of a 96-well tissue culture plate with a lid (Falcon, Oxnard, CA). Eight plates were prepared for the testing of each drug combination. Each plate had one 8-well control column containing medium alone and one 8-well control column containing cells but no drugs. Drug solutions of STI571 and other drugs at different concentrations were then added (50 µL) to 8 wells containing cell suspensions. Because daily administration of STI571 has been used in clinical settings, KU812, K562, and TCC-S cells were simultaneously and continuously exposed to the drugs for 5 days. TCC-Y cells, which were very small and had a longer doubling time than other cell lines, were incubated with the drugs for 7 days.MTT assay Viable cell growth was determined by MTT reduction assay as described previously.23 For the background control, control (no drug), each drug or drug combination, the 4 intermediate data values among the 8 data values were used for the analysis and the 2 highest and the 2 lowest values were discarded. Hydroxyurea influenced the absorbance at 570 nm and erroneously high MTT values were observed. Thus, each concentration of hydroxyurea was also added to the respective background control for the study of STI571-hydroxyurea combinations. For all cell lines examined, we established a linear relation between the MTT assay value and the cell number within the range of the experiments shown.Analysis of the evaluation of the cytotoxic effects of STI571 with the other agents The cytotoxic interactions of STI571 with other agents at the point of IC80 were evaluated by isobologram (Steel and Peckham).24 The IC80 was defined as the concentration of drug that produced 80% cell growth inhibition, that is, 80% reduction of absorbance. We used the IC80 value instead of the more common IC50 value, because IC80 would be more important than IC50 for the evaluation as anticancer agents. Although the drug interaction at IC90 or more would be more important than IC50 or IC80, it is difficult to obtain reliable data at the IC90 or greater using MTT assay.The isobologram of Steel and Peckham The theoretical basis of the isobologram method and the procedure for making isobolograms have previously been described in detail.24-26 Based on the dose-response curves of STI571 and the other agents, 3 isoeffect curves were constructed (Figure 1). If the agents act additively by independent mechanisms, combined data points will lie near the mode I line (hetero-addition). If the agents act additively by similar mechanisms, combined data points will lie near the mode II lines (iso-addition).
Recent studies suggested that many anticancer agents kill tumor cells by apoptosis. The initiating signals to apoptosis by anticancer agents are still poorly understood. In addition, numerous converging pathways integrate the initiating signals and transmit the results to mitochondria causing activation of caspase cascade. Because it is unknown in advance whether the combined effects of 2 agents will be hetero-additive, iso-additive, or an effect intermediate between these extremes, all possibilities should be considered. Thus, when the data points of the drug combination fell within the area surrounded by 3 lines (envelope of additivity), the combination was regarded as additive. The envelope of additivity should not be considered as a reliable definition of additivity. An expression of the uncertainty is an important concept of the isobologram method of Steel and Peckham. When the data points fell to the left of the envelope, that is, the combined effect was caused by lower doses of the 2 agents than was predicted, the drug combination was regarded as having a supra-additive effect (synergism). When the points fell to the right of the envelope, that is, the combined effect was caused by higher doses of the 2 agents than was predicted, but within the square or on the line of the square, the combination was regarded as having a subadditive effect; that is, the combination was superior or equal to a single agent but was less than the additive effect. When the data points were outside the square, the combination was regarded as having a protective effect, that is, the combination was inferior in cytotoxic action to a single agent. Both subadditive and protective interactions were regarded as antagonism. Data analysis When the observed data points in combination mainly fell within the envelope of additivity, the combination was considered as having an additive effect. The mean values of the observed data were compared with those of the predicted maximum values and those of the predicted minimum values for an additive effect.27 If the mean values of the observed data were equal to or smaller than those of the predicted maximum values and equal to or larger than those of the predicted minimum values, the combination was regarded as an additive effect.When the observed data points in the combinations mainly fell in the area of supra-additivity or in the areas of subadditivity and protection, that is, the mean value of the observed data were smaller than those of the predicted minimum values or larger than those of the predicted maximum values, the combinations were considered to have a synergistic or antagonistic effect, respectively. To determine whether the condition of synergism (or antagonism) truly existed, a statistical analysis was performed. The Wilcoxon signed-rank test was used to compare the observed data with the predicted minimum (or maximum) values for additive effects, which were closest to the observed data (ie, the data on the boundary [mode I or mode II lines] between the additive area and supra-additive area [or subadditive and protective areas]).26 Combinations with significant values (P < .05) were defined as having synergistic effects (or antagonistic effects), and those with insignificant values (P > .05) were defined as having additive to synergistic effects (or additive to antagonistic effects). All statistical analyses were performed using the StatView 4.01 software program (Abacus Concepts, Berkeley, CA).
Cytotoxic effects of STI571 and other agents against Ph+ leukemia cells The dose-response curves of STI571 for KU812, K-562, TCC-S, and TCC-Y are shown in Figure 2. The IC80 values of STI571 for these cells were 84 ± 9 nM, 247 ± 30 nM, 72 ± 6 nM, and 510 ± 80 nM, respectively (Figure 2). Nine human Ph leukemia cell lines (HL-60, KG-1, U937,
MOLT-3, SALT-3, Raji, HBL, JOK, SKW-3), and 4 solid tumor cell lines
(A549, MCF-7, PA-1, WiDr) tested were resistant to STI571 and 10 µM
STI571 had almost no effects on the growth inhibition of these cell
lines (data not shown). Table 1
summarizes the IC80 levels of Ph+ leukemia cell
lines to the antileukemic agents used in the present study.
Cytotoxic interaction between STI571 and other agents Figure 3A-D shows the dose-response curves for STI571 in combination with recombinant IFN- , hydroxyurea,
methotrexate, or vincristine in the KU812 cells. Each isobologram was
generated based on such dose-response curves.
Cytotoxic interaction between STI571 and IFNs- , whereas TCC-S
cells were relatively resistant to IFN-. Thus, the effects of the
drug combination at IC40 were used for TCC-S cells. K-562 cells were highly resistant to IFN- and could not be used for the
study. Figure 4A-C shows the isobolograms
of STI571 in combination with recombinant IFN- against KU812, TCC-S,
and TCC-Y cells. In the KU812 and TCC-Y cells, all or most of the data
points for the combination fell to the left of the envelope of
additivity. The mean values of the observed data (KU812: 0.27, and
TCC-Y: 0.16) were smaller than those of the predicted minimum additive values (0.40 and 0.32, respectively; Table
2). The observed data and the predicted
minimum values were compared by Wilcoxon signed-rank test. The observed
data in the KU812 and TCC-Y cells were significantly smaller than the
predicted minimum values (P < .01 and
P < .02, respectively), indicating a synergistic effect
of the simultaneous exposure to these 2 agents (Table 2). The observed
data (0.29) in the TCC-S cells were not significantly smaller than the
predicted minimum values (0.34) (P > .05), indicating
additive to synergistic effects of the simultaneous exposure to these 2 agents (Table 2). For the combination of STI571 with natural IFN-
against KU812, TCC-Y, and TCC-S cells, all or most of the data points for the combination fell to the left of the envelope of additivity (isobologram not shown). The observed data in the KU812, TCC-S, and
TCC-Y cells were significantly smaller than the predicted minimum
values, indicating a synergistic effect of the simultaneous exposure to
these 2 agents (Table 2).
Cytotoxic interaction between STI571 and hydroxyurea Figure 5A-D shows the isobolograms of this combination in the KU812, K-562, TCC-S, and TCC-Y cells. In the KU812 cells, the combined data points fell within the envelope of additivity (Figure 5A). The mean value of the data (0.61) was larger than that of the predicted minimum values (0.42) and smaller than that of the predicted maximum values for an additive effect (0.73), indicating that the simultaneous exposure to STI571 and hydroxyurea produced an additive effect (Table 2). Similarly, in the K562, TCC-S, and TCC-Y cells, all or most of the data points for the combination fell within the envelope of additivity (Figure 5B-D). The mean values of the observed data were between those of the predicted minimum values and those of the predicted maximum values for an additive effect, indicating an additive effect of the simultaneous exposure to these 2 agents in all 4 cell lines (Table 2).
Cytotoxic interaction of STI571 in combination with cytarabine, homoharringtonine, doxorubicin, or etoposide In all 4 cell lines, all or most of the data points for the combination fell within the envelope of additivity (isobologram not shown). The mean values of the data were larger than those of the predicted minimum values and smaller than those of the predicted maximum values for an additive effect, indicating that the simultaneous exposure to STI571 with cytarabine, homoharringtonine, doxorubicin, or etoposide produced additive effects in all 4 cell lines (Table 2).Cytotoxic interaction between STI571 and 4-hydroperoxy-cyclophosphamide In the KU812, TCC-S, and TCC-Y cells, all or most of the combined data points fell within the envelope of additivity (isobologram not shown). The mean values of the data were between those of the predicted minimum values and those of the predicted maximum values for an additive effect, indicating that the simultaneous exposure to STI571 and 4-hydroperoxy-cyclophosphamide produced additive effects (Table 2). In the K562 cells, the data points for the combination fell within the envelope of additivity and in the area of supra-additivity (isobologram not shown). The mean values of the data were not significantly smaller than those of the predicted minimum values, indicating additive to synergistic effects (Table 2).Cytotoxic interaction between STI571 and methotrexate Figure 6A-D shows the isobolograms of this combination. In the KU812, TCC-S, and TCC-Y cells, all or most of the data points for the combination fell in the areas of subadditivity and protection. The mean values of the observed data were larger than those of the predicted maximum additive values. The observed data were significantly higher than the predicted maximum values, indicating antagonistic effects of the simultaneous exposure to these 2 agents (Table 2). In the K562 cells, the data points fell within the envelope of additivity and in the areas of subadditivity and protection. The observed data were not significantly higher than the predicted maximum values, indicating additive to antagonistic effects of the simultaneous exposure to these 2 agents (Table 2).
Cytotoxic interaction between STI571 and vincristine Figure 7A-D shows the isobolograms of this combination. In all 4 cell lines, the data points for the combination fell within the envelope of additivity and in the area of supra-additivity. The mean values of the observed data were smaller than those of the predicted minimum additive values. In the KU812, TCC-S, and TCC-Y cells, the observed data were significantly smaller than the predicted minimum values, indicating synergistic effects of the simultaneous exposure to STI571 and vincristine (Table 2). In the K562 cells, the observed data were not significantly smaller than the predicted maximum value, indicating additive to synergistic effects of the simultaneous exposure to these 2 agents (Table 2).
We have also made the isobolograms of STI571 in combination with the used agents at the IC50 level (data not shown). Basically, similar effects were observed for all combinations in all 4 cell lines.
STI571 is a new antileukemic agent targeting BCR/ABL tyrosine kinase.14 A preliminary clinical study has shown that STI571 has significant activity in patients with chronic phase CML in whom IFN therapy has failed.18 STI571 induced complete hematologic remission in 96% after at least 4 weeks of therapy and cytogenetic remission in 33% of patients within 2 months of therapy. Side effects were mild and no dose-limiting toxicity has been encountered. The purpose of the present study was to investigate the appropriate combinations of STI571 with antileukemic agents against 4 human Ph+ leukemia cell lines at the cellular level. When the dose-response curves are far from linear, as is usually the case in cancer chemotherapy and was also the case in this study, the nature of an additive response has been controversial.29 We used the Steel and Peckham isobologram method to evaluate the cytotoxic interaction between STI571 and other agents. Although a large number of data points are required, this method can be used to calculate the additive interaction of any combination, irrespective of the shapes of the dose-response curves of the agents and of whether they have independent or overlapping damage. Currently, the most commonly used agent for CML during the chronic
phase is IFN- In the present study, we observed that STI571 produced synergistic or
additive to synergistic effects with recombinant and natural IFNs- There have been several conflicting experimental findings for the
combination of STI571 and IFN- Hydroxyurea or cytarabine alone or in combination with other agents
have also been used for patients with Ph+ and
Ph There have been 2 experimental findings for the combinations of STI571 and hydroxyurea or cytarabine. Thiesing and coworkers reported that the combination of STI571 and hydroxyurea showed less than an additive effect, whereas the combination of STI571 and cytarabine showed synergistic effects against K562 and MO7p210 cells.20 Topaly and colleagues reported that the combinations of STI571 and hydroxyurea or cytarabine showed synergistic effects in Ph+ BV173 and EM-3 cells using the median effect principle.21 The present findings are different from their findings, probably due to different experimental conditions and analytical methods. As described previously, the isobologram of Steel and Peckham is stricter for synergism and antagonism. Homoharringtonine, an alkaloid extracted from the evergreen
Ceplalotaxus harringtonia, is a new anticancer
agent that is currently incorporated into the combination protocol for
CML in chronic phase and AML.8,33 The inhibition of
protein synthesis is considered the major cytotoxic mechanism of this
agent.34 A recent clinical study showed that
homoharringtonine induced complete hematologic remission in 92% and
cytogenetic remission in 60% of patients with early
CML.35 These findings were significantly better than those
observed for IFN- We further studied the effects of STI571 in combination with doxorubicin, etoposide, 4-hydroperoxy-cyclophosphamide, methotrexate, and vincristine, which are commonly used for the treatment of CML in blastic crisis and Ph+ ALL. Although the clinical activity of STI571 against CML blastic crisis and Ph+ ALL is not yet clear, Ph+ leukemia cell lines, most of which were sensitive to STI571, were established from patients with CML blastic crisis and Ph+ ALL. This suggests that STI571 would have an activity against these Ph+ leukemias. Simultaneous exposure to STI571 and doxorubicin, etoposide, or 4-hydroperoxy-cyclophosphamide produced additive effects in all 4 or 3 of 4 cell lines. These findings suggest that the simultaneous administration of STI571 with doxorubicin, etoposide, or cyclophosphamide would have the expected activity and would be favorable for the treatment of Ph+ leukemias. 4-Hydroperoxy-cyclophosphamide has been used for purging leukemia cells in vitro.36 STI571 is also considered as an agent for purging Ph+ leukemia cells in vitro. Simultaneous exposure to these 2 agents in vitro appears appropriate for purging Ph+ leukemia cells. Simultaneous exposure to STI571 and methotrexate produced antagonistic or additive to antagonistic effects in all 4 cell lines. The simultaneous administration of STI571 and methotrexate would be therefore inadequate. The mechanisms of the antagonistic effects of STI571 with methotrexate are unknown. Because methotrexate is a highly cell cycle-specific agent, cell cycle disturbances by STI571 might interfere with the action of methotrexate. Simultaneous exposure to methotrexate and most anticancer agents was also observed to produce antagonistic effects in our previous study (unpublished data). Simultaneous exposure to STI571 and vincristine produced synergistic or additive to synergistic effects in all 4 cell lines, suggesting that the simultaneous administration of STI571 and vincristine would be highly active against CML in lymphoid crisis and Ph+ ALL. In our previous analysis on the combination of vinca alkaloids with a variety of anticancer agents, no drugs produced synergistic effects with vincristine during simultaneous exposure25 (and unpublished data). The synergism between STI571 and vincristine is therefore exceptional and should draw special attention. Mitotic inhibitors such as vinca alkaloids and taxanes have been shown to induce phosphorylation of bcl-2 family proteins including Bcl-2 and Bcl-x, which appears to be accompanied by the loss of function, and to induce apoptosis.37,38 Recently, Oetzel and colleagues39 reported that STI571 induced apoptosis in BCR/ABL-transfected cell lines by down-regulation of Bcl-x. The synergistic cytotoxicity of STI571 and vincristine may be attributable to their effects on Bcl-x. These findings may be of importance in the design of STI571-based combination chemotherapy. In the present study, however, we used only simultaneous and continuous exposure to STI571 and other agents for 5 or 7 days. The results obtained on combining STI571 and other agents after 1 or 24 hours of exposure may be different from those obtained after a 5- or 7-day exposure. The order in which STI571 and other agents are administered to cells may also play a role. In addition, there are a number of difficulties in the translation of findings from in vitro to clinical therapy. The pharmacokinetics of STI571 and other agents are significantly different between them. Furthermore, the pharmacokinetic interaction and the toxic effects of the combinations cannot be measured in vitro. In addition, the cell kinetics and cell biochemistry may be quite different. Finally, the administration schedules in the present study were not always similar to those of clinical settings. These differences between in vitro and clinical systems would influence the cytotoxic interaction of STI571 and other agents. In conclusion, the present study showed that simultaneous exposure to
STI571 and IFN-
Submitted July 19, 2000; accepted November 21, 2000.
Supported by a Grant-in-Aid for Cancer Research (11-8) from the Ministry of Health and Welfare, Japan.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Yasuhiko Kano, Division of Medical Oncology, Tochigi Cancer Center, 4-9-13 Yonan, Utsunomiya, Tochigi, 320-0834, Japan; e-mail: ykano{at}tcc.pref.tochigi.jp.
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C. Yu, G. Krystal, L. Varticovksi, R. McKinstry, M. Rahmani, P. Dent, and S. Grant Pharmacologic Mitogen-activated Protein/Extracellular Signal-regulated Kinase Kinase/Mitogen-activated Protein Kinase Inhibitors Interact Synergistically with STI571 to Induce Apoptosis in Bcr/Abl-expressing Human Leukemia Cells Cancer Res., January 1, 2002; 62(1): 188 - 199. [Abstract] [Full Text] [PDF]
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S. M. Graham, H. G. Jorgensen, E. Allan, C. Pearson, M. J. Alcorn, L. Richmond, and T. L. Holyoake Primitive, quiescent, Philadelphia-positive stem cells from patients with chronic myeloid leukemia are insensitive to STI571 in vitro Blood, January 1, 2002; 99(1): 319 - 325. [Abstract] [Full Text] [PDF]
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Y. A. Elsayed and E. A. Sausville Selected Novel Anticancer Treatments Targeting Cell Signaling Proteins Oncologist, December 1, 2001; 6(6): 517 - 537. [Abstract] [Full Text] [PDF]
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B. J. Druker, C. L. Sawyers, R. Capdeville, J. M. Ford, M. Baccarani, and J. M. Goldman Chronic Myelogenous Leukemia Hematology, January 1, 2001; 2001(1): 87 - 112. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
80°C. Appropriate drug concentrations were made
by dilution with fresh medium immediately before each experiment. The
final concentration of dimethyl sulfoxide in the media was less than
0.1%, and it had no effect on the cell growth inhibition in the
present study.
), 10 (
), 20 (
), 30 (
), 40 (
), 50 (
), 60 (
), 80 (
) and 100 (x) nM. The concentrations of the combined drugs are
shown on the abscissa. Each point represents the mean value for at
least 3 independent experiments; the SEMs were less than 20% and
were omitted.
perspectives on an active new natural product.
J Clin Oncol.
1986;4:1563-1568