Japanese Journal of Clinical Oncology 30:562-567 (2000)
© 2000 Foundation for Promotion of Cancer Research
Comparison of In Vitro Growth-inhibitory Activity of Carboplatin and Cisplatin on Leukemic Cells and Hematopoietic Progenitors: the Myelosuppressive Activity of Carboplatin May Be Greater Than Its Antileukemic Effect
1Division of Hematology/Oncology, Departments of Internal Medicine and 2Pediatrics, National Cheng Kung University Hospital, Tainan, Taiwan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentREFERENCES |
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Background: Carboplatin [cis-diammine(cyclobutane-1,1-dicarboxylato)platinum(II)] has been shown to be an active agent for acute myeloid leukemia. This second-generation platinum drug has less nephrotoxicity and ototoxicity but more myelotoxicity than does the first-generation platinum drug cisplatin. The study was designed to elucidate whether their myelosuppressive activities equal their antileukemic effects.
Methods: Cisplatin and carboplatin were used to treat four leukemic cell lines (CEM, HL60, K562 and U937), blast cells from 10 leukemic patients and hematopoietic progenitors from five umbilical cord blood samples.
Results: The mean IC50 of leukemic cell lines was 0.4 and 6.2 µg/ml, the mean IC50 of patients’ leukemic blasts was 2.0 and 22.4 µg/ml and the mean IC50 of hematopoietic progenitors (BFU-E, CFU-E and CFU-GM) was 1.8 and 1.7 µg/ml for cisplatin and carboplatin, respectively.
Conclusions: Carboplatin required a 10 times higher drug concentration than cisplatin to induce a similar degree of growth inhibition on leukemic cells. However, the hematopoietic progenitors responded equally to cisplatin and carboplatin at the same drug concentration. The results suggest that the myelosuppressive activity of carboplatin is greater than its antileukemic effect.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentREFERENCES |
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Although the treatment of childhood leukemia has achieved great success, with an average 85–95% complete response (CR) rate and 80–90% long-term survival; the results for adult leukemic patients are still unsatisfactory: the average CR rate is 70–80% but long-term survival is only 20–30% (1). The failure of the treatment is mainly caused by the development of cellular drug resistance (2). Therefore, the discovery of better antileukemic drugs is essential.
Carboplatin [cis-diammine(cyclobutane-1,1-dicarboxylato) platinum(II)] is a second-generation platinum complex. Unlike the first-generation platinum drugs, such as cisplatin, carboplatin has less nephrotoxicity and ototoxicity but has substantial, adverse myelosuppressive effects (3). Because of this toxicity, carboplatin has been used in treating hematological malignancies. Some studies have demonstrated that carboplatin as a single agent or in combination with other cytotoxic drugs is active in treating acute myeloblastic leukemia (AML) or blast crisis of chronic myeloid leukemia (CML) (4–6). However, the assumption that a drug with higher myelosuppressive activity should have a stronger antileukemic effect has never been clearly proved. Since the anti-neoplastic activity of carboplatin, in treating solid tumors, was shown to be inferior to that of cisplatin in several cancers (3,7), it is desirable to know the cytotoxic activities of carboplatin and cisplatin on hematopoietic progenitors and leukemic cells. Our results suggest that the myelosuppressive activity of carboplatin is not the same as its antileukemic effect.
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MATERIALS AND METHODS |
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Leukemic Cells from Patients
Leukemic cells were obtained from bone marrow or peripheral blood samples which were taken from patients for routine diagnostic procedures. There were seven patients with acute myeloblastic leukemia (AML) and three with acute lymphoblastic leukemia (ALL) according to the FAB diagnostic classification. To isolate mononuclear cells, the samples were diluted twofold in phosphate-buffered saline (PBS), laid on top of an equal volume of Ficoll-Hypaque (1.077 g/cm2) (Pharmacia, Uppsala, Sweden) and centrifuged for 25 min at 500 g. The cells were harvested and frozen in 90% fetal calf serum (FCS) (Gibco, New York, USA) with 10% dimethyl sulfoxide (DMSO) (Sigma, St. Louis, MO, USA). Before the study, the cells were thawed and cultured overnight in RPMI 1640 medium (Gibco) containing 15% FCS, 100 IU/ml penicillin, 100 µg/ml streptomycin, 0.125 µg/ml fungizone, 200 µg/ml gentamycin, 2 mmol/l L-glutamine (Sigma), 5 µg/ml insulin, 5 µg/ml transferrin and 5 ng/ml sodium selenite (ITS was obtained from Sigma) (8).
Leukemic Cell Lines
Four human leukemic cell lines, CEM, HL60, K562 and U937, were used. They were developed from T-acute lymphoblastic leukemia, acute promyelocytic leukemia, chronic myelogenous leukemia in blastic transformation and promonocytic leukemia, respectively. Cells were propagated in RPMI 1640 medium supplemented with 10% heat-inactivated FCS, penicillin 100 IU/ml and streptomycin 100 µg/ml in a highly humidified atmosphere of 5% CO2 at 37°C.
Hematopoietic Progenitor Cells
Volumes of 10–15 ml of heparinized umbilical cord blood were obtained from five normal deliveries. Mononuclear cells were seperated by Ficoll-Hypaque density centrifugation at 500 g for 25 min. The cells at the interface were collected and washed twice before study.
MTT Colorimetric Assay
Leukemic cells from patients were diluted with medium to 1 x 106 cells/ml and aliquots (1.8 x 105cells/180 µl) were placed in individual wells in 96-well multiplates. In the studies of leukemic cell lines, 5 x 103 cells/180µl were added to the individual well. Each well in addition received 20 µl of either cisplatin (Farmitalia, Barcelona, Spain) or carboplatin (Bristol-Myers, Madrid, Spain), which had been serially diluted 10-fold in distilled water. All six wells in the same column had an identical concentration of cisplatin, ranging from 0.001 to 100 µg/ml, or carboplatin, ranging from 0.01 to 1000 µg/ml. Cells were incubated for 3 days, then their viability was determined by the colorimetric MTT [3-(4,5-dimethylthiazol-2-yl)-5-diphenyltetrazolium bromide; Sigma] assay (9). The MTT solution (5 mg/ml in PBS, 20 µl) was added to each well and the plates were incubated for 4 h. The bulk of the medium from each well was suctioned off, leaving 10–20 µl behind, together with purple formazan crystals. DMSO (200 µl) was added to each well to dissolve the crystals and the plates were shaken for 10 min on a shaker. The absorbance of each well was read at 540 nm on a Titertek MCC 340 plate reader.
Clonogenic Assay for Leukemic Cell Lines
Cells (1x104) from the four leukemic cell lines were cultured in 3 ml of 1% methylcellulose supplemented with RPMI 1640 medium and 30% FCS for 7 days. Colonies consisting of more than 40 cells were counted using a dissection microscope.
Clonogenic Assay for Hematopoietic Progenitor Cells (10)
BFU-E
Mononuclear cord blood cells were cultured in 0.9% methylcellulose, supplemented with 5% Iscove’s modified Dulbecco’s medium (IMDM) (Gibco), 30% FCS, 2 units/ml of recombinant human erythropoietin (rh-EPO) (Cilag, Zug, Switzerland), 1% bovine serum albumin (BSA) (Gibco), 10–4 M 2-mercaptoethanol (2-ME) (Sigma) and 5% phytohemagglutinin–leukocyte-conditioned medium (PHA-LCM) (PHA from Gibco). The final concentration of cells was 2x105/ml. Cisplatin and carboplatin were added to the culture medium at final concentrations of 0, 0.1, 1, 5, 10 and 100 µg/ml. Colonies consisting of more than 40 cells were scored on day 14 using a dissection microscope.
CFU-E
The procedures and culture conditions were similar to those in the assay for BFU-E, except that 0.9% methylcellulose was supplemented with 10% instead of 5% IMDM and without PHA-LCM.
CFU-GM
Mononuclear cells from cord blood were cultured in 0.3% agar, supplemented with 10% IMDM, 20% FCS, 10–4 M 2-ME and 2 ng/ml of granulocyte-macrophage colony-stimulating factor (GM-CSF) (Sandoz, Hong Kong). The final concentration of cells was 2 x 105/ml. The concentrations of drugs were the same as above. Colonies were scored on day 14.
Comparison of Drug Activity
The 50% inhibitory concentration (IC50) of the MTT assay was calculated from the percentage of the control absorbance. The percentage survival of a colony was calculated from the equation
The mean IC50 values for cisplatin and carboplatin in leukemic cell lines, leukemic cells from patients and hematopoietic progenitor cells were compared and analyzed by a two-tailed Wilcoxon signed-rank test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentREFERENCES |
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Leukemic Cells from Patients
Most of the frozen leukemic cells were successfully cultured in vitro and the percentage of leukemic blasts in each sample was above 50% after purification by Ficoll-Hypaque density-gradient centrifugation. Table 1 summarizes the characteristics of patients, source of samples, the percentage of blasts following purification and the IC50s of leukemic cells for cisplatin and carboplatin. Although there was a wide range of variation in cellular sensitivity to the two drugs, the magnitude of the difference between sensitivities to cisplatin and carboplatin in each sample was constantly around 10-fold. The mean IC50 of the 10 samples treated with cisplatin and carboplatin was 2.0 and 22.4 µg/ml, respectively, which again demonstrated a 10-fold difference in antileukemic activity between the two platinum derivatives.
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Leukemic Cell Lines
Four leukemic cell lines (CEM, HL60, U937 and K562) were used and their sensitivities to cisplatin and carboplatin were compared with the results from patients’ samples. As shown in Table 2, the four leukemic cell lines are more sensitive to cisplatin than to carboplatin. The mean IC50 of the four cell lines, evaluated by MTT assay, was 0.4 and 6.2 µg/ml for cisplatin and carboplatin, respectively. The 15-fold difference between their sensitivities to cisplatin and carboplatin is compatible with the findings using leukemic cells from patients. To study the longer survival potential of the leukemic cells after treatment with platinum derivatives, clonogenic assays were performed. The results of clonogenic assay were similar to those from MTT assay, with mean IC50s of 0.5 and 4.7 µg/ml for cisplatin and carboplatin, respectively. The sensitivities of leukemic cell lines to cisplatin and carboplatin were not affected by the different measuring methods.
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Hematopoietic Progenitor Cells
The actual numbers of colonies of hematopoietic progenitors after treatment with various concentrations of cisplatin and carboplatin are listed in Table 3, which shows that the formation of hematopoietic progenitor colonies, identified as BFU-E, CFU-E and CFU-GM, was inhibited by cisplatin and carboplatin at low dose levels (Table 4). The sensitivities of these three progenitors to platinum derivatives were very close, so we analyzed them together. The mean IC50 of the three hematopoietic progenitors for cisplatin and carboplatin was 1.8 and 1.7 µg/ml, respectively, which indicated that the progenitors of erythroid cells, granulocytes and macrophages are equally sensitive to cisplatin and carboplatin.
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By two-tailed Wilcoxon signed-rank statistical analysis, the difference between the mean IC50s of leukemic cells from patients and of four leukemic cell lines, treated with cisplatin and carboplatin, was statistically significant (p < 0.05), but was not significant for hematopoietic progenitors treated with cisplatin and carboplatin.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentREFERENCES |
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The mean IC50 of cisplatin for leukemic cells from patients and hematopoietic progenitors was 2.0 and 1.8 µg/ml, respectively, and the mean IC50 of carboplatin for leukemic cells from patients and hematopoietic progenitors was 22.4 and 1.7 µg/ml, respectively. When cisplatin was given as an intravenous bolus injection at a dosage of 100 mg/m2, a peak plasma level of ~6 µg/ml was reached immediately and decreased to <2 µg/ml within 2 h (11). Similar administration of 375 mg/m2 of carboplatin resulted in a peak plasma level of 39 µg/ml, which declined to ~9 µg/ml within 2 h (12). Therefore, 2 h after intravenous bolus injection of the above dose of cisplatin, the drug level in plasma is still sufficient to inhibit 50% growth of leukemic and hematopoietic cells. However, 9 µg/ml of carboplatin in plasma, although still very toxic to hematopoietic cells, is inadequate to inhibit growth of leukemic cells. The major reason for the different cytotoxicity between cisplatin and carboplatin has been attributed to their aquation rates which then influence the kinetics of their interaction with DNA (13). The rate constant for the aquation of carboplatin is 100-fold lower than that for cisplatin (13,14). Micetich et al. found that the peak level of DNA interstrand cross-links in cells treated with carboplatin for 2 h occurs 6–12 h later than that for cells treated with cisplatin for the same duration (15). In a study using one ovarian cell line, exposure of cells to carboplatin had to be extended to 24 h in order to achieve a cytotoxicity equal to that induced by 1 h of exposure to cisplatin at the same concentration of 1 µg/ml (16). To overcome the problem of slow aquation rate, administration of carboplatin was changed to continuous intravenous injection over 3–5 days in the treatment of leukemia (4–6). In the present study, cisplatin and carboplatin were added to the culture medium for more than 3 days, which allowed adequate aquation of both drugs. Therefore, the observed differences in sensitivity of cells to cisplatin and carboplatin were not related to their different aquation rates.
Since the cellular viability of leukemic cells from patients after treatment with drugs was evaluated by MTT assays, whereas the growth of hematopoietic progenitors after treatment was measured by clonogenic assays, to exclude the possibility that differences in cellular sensitivity to drugs observed in the study was due to the use of different assay systems, we investigated the cytotoxic activities of drugs on leukemic cell lines by MTT and colony-forming assays. The sensitivities of four leukemic cell lines to platinum derivatives measured by the two different assays were similar, which indicates that the cellular sensitivities to cisplatin and carboplatin are independent of the assay technique.
Data from our experiments show that carboplatin requires a 10 times higher drug concentration than cisplatin to induce a similar degree of growth inhibition of leukemic cells. This observation is consistent with reports by other investigators that carboplatin is ~10–40 times less potent than cisplatin in growth inhibition of various cell lines (17–19). Regarding DNA level, Hongo et al. have shown that carboplatin requires a 10 times higher drug concentration and 7.5 times longer incubation time than cisplatin to induce the same degree of conformational change on pUC18 plasmid DNA (20). Taken together, we may say that carboplatin, after an adequate time for aquation, requires a 10-fold higher drug concentration than cisplatin to induce a similar degree of DNA damage and tumor cell growth inhibition. However, our findings that carboplatin inhibited the growth of hematopoietic progenitors as effectively as cisplatin at the same drug level suggests that carboplatin has a stronger anti-proliferative activity on hematopoietic cells than on leukemic cells. The mechanism by which hematopoietic cells are more sensitive than leukemic cells to carboplatin is not yet clear. However, cell lines that are more sensitive to carboplatin than cisplatin have been recognized (21). Clinically, carboplatin can be substituted for cisplatin in the treatment of ovarian cancer, non-small-cell and extensive-stage small-cell lung cancers, but is inferior to cisplatin in germ cell, head and neck and esophageal cancer (3). The above data suggest that various type of cells may respond differently to cisplatin and carboplatin. Moreover, our findings are consistent with the results from a murine model, in which carboplatin was shown to be more toxic than cisplatin for hematopoietic stem cells and was less active than cisplatin against the mouse leukemia L1210 (22). In treating leukemic patients, carboplatin has already been shown to be an effective agent. With its modest non-hematological toxicity, carboplatin should be considered the best choice among platinum derivatives for leukemia therapy. However, cisplatin may have a role in the control of leukemia under certain circumstances. For example, a second identical course of chemotherapy is administered to patients by some investigators (23) if residual leukemic blasts are found in the day-14 bone marrow during the induction chemotherapy for acute leukemia. Although the added chemotherapy may enhance the clearance of blast cells, it will also lengthen the cytopenic duration and increase treatment morbidities owing to its prominent myelosuppressive activity. In this condition, an agent with active cytotoxicity to leukemic blasts but less toxic to hematopoietic progenitors, such as cisplatin, may be a good substitute for the second course of chemotherapy.
This study has demonstrated that the myelosuppressive activity of carboplatin is greater than its antileukemic effect. With a better understanding of the anti-proliferative activities of platinum analogs, we may apply these drugs more effectively in the treatment of leukemia.
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Acknowledgment |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentREFERENCES |
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This work was supported by National Cheng Kung University Hospital grants (NCKUH 84-004 and 87-004).
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FOOTNOTES |
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+ For reprints and all correspondence: Wu-Chou Su, Division of Hematology/Oncology, Department of Internal Medicine, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, 138 Sheng-Li Road, Tainan 70428, Taiwan. E-mail: sunnysu@mail.ncku.edu.tw
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Received June 30, 2000; accepted September 19, 2000.
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