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Ether lipids are effective cytotoxic drugs against multidrug-resistant acute leukemia cells and can act by the induction of apoptosis
Leukemia Research Vol. 21, No. 1, pp. 31-43, 1997. Cmvrieht 8 1997 Elsevia Science Ltd. All rights reserved Printed in &eat Britain 014s2126/97 $17.00 + o.cKl
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ETHER LIPIDS ARE EFFECTIVE CYTOTOXIC DRUGS AGAINST MULTIDRUG-RESISTANT ACUTE LEUKEMIA CELLS AND CAN ACT BY THE INDUCTION OF APOPTOSIS Leo F. Verdonck and Hans G. van Heugten Department of Haematology, University Hospital, Utrecht, The Netherlands (Received 19 June 1995. Revision accepted 20 April 1996) Abstract-We studied the cytotoxic effects of two ether lipids, a relatively new class of anticancer drugs, on multidrug-resistant (MDRI) leukemia cells of patients with acute leukemias who had failed induction treatment. The cytotoxicity of ether lipids was determined by the elimination of clonogenic leukemia cells from leukemic cultures or, in case of failure to generate leukemic cultures, by the inhibition of 3[Hlthymidine incorporation in leukemic blasts. At dose levels of 50 pg/ml, a plasma level that can be achieved after oral intake, the MDRIpositive blasts were killed, both of patients with drug-resistant acute myeloid leukemia (AML) and of patients with drug-resistant acute lymphoblastic leukemia (ALL). The leukemic blasts were killed by the induction of apoptosis. These data suggest that ether lipids may be effective antileukemic drugs and that their cytotoxic function is not affected by MDRI. 0 1997 Elsevier Science Ltd. Key words: Ether lipid, acute leukemia,
multidrug
resistance,
apoptosis.
[ll-161. Probably more attractive, however, will be the development of drugs that are not affected by MDRl. Previously, we and others have demonstrated that the ether lipid ET-18-OCH3 (ALP), a synthetic analog of 2lysophosphatidylcholine, exhibits selective antineoplastic activity to leukemic cells [ 17-201. This was followed by studies exploring the possibility of ALP (or its analogs) being an agent for purging bone marrow grafts of patients with acute leukemias for autologous marrow transplantation [19-231. Another ether lipid, hexadecylphosphocholine (miltefosine) has been investigated recently in clinical phase I/II studies as an oral anticancer drug for patients with solid tumors [24,25]. The mechanisms by which ether lipids exert their cytotoxic effects to malignant cells are under extensive investigation and several hypotheses have been proposed [17,18, 26-311. Here we report the results of in vitro studies showing that ALP and miltefosine effectively kill multidrugresistant blasts from patients with refractory acute leukemias and can act by the induction of apoptosis.
Introduction Although many patients with acute leukemias will achieve a complete remission with combination chemotherapy, the majority of patients eventually relapse because of the emergence or expansion of drug-resistant leukemic cells. Furthermore, some patients already have drug-resistant disease at presentation. Several mechanisms are recognized by which tumor cells become resistant to anticancer drugs [l]. Among these, the multidrug resistance gene (MDRl) encoding the transmembrane protein P-glycoprotein (Pgp), has been the subject of the majority of investigations [2-51. Pgp acts as a drug efflux pump, capable of expelling a variety of anticancer drugs from the cytosol [l-5]. Overexpression of MDRl results in an increased activity of Pgp and has been associated with drug resistance in patients with acute leukemias [6-lo]. This has led to the introduction of pharmacological interventions, e.g. the use of cyclosporine, which can competitively inhibit Pgp Abbreviations: ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; L-CFCs, leukemic colony-forming cells; MDRl, multidrug-resistant; Pgp, P-glycoprotein. Correspondence to: Leo F. Verdonck, MD, PhD, University Hospital Utrecht, Departmentof Haematology(G03.647),P.O. Box 85.500, 3508 GA Utrecht, The Netherlands (Tel: +31 30 50 72 30; Fax: +31 30 51 18 93; E-mail: [email protected]).
Materials and Methods Patients
Ten patients with acute leukemias were studied. The characteristics of the patients are given in Table 1. Four 37
38
L. F. VerdonckandH. G. van Heugten Table 1. Patient characteristics
Patient number 1 2 3 4 5 6 7 8 9 10
Sex/age
Diagnosis
M/39 M/33 M/32 Ml33 M/48 Ml46 M/32 M/25 F/23 F/44
AML, Ml AML, M4 AML, Ml AML, M2 S-AML, Ml S-AML, M2 S-AML, Ml C-ALL T-ALL (1st relapse) T-ALL (3rd relapse)
Responseto Induction treatment 2nd line treatment NR NR NR NR NR NR NR NR NR NT
CR CR NR NR NR NT CR NR NR NT
Abbreviations: AML, acute myeloblastic leukemia; FAH Classification Ml, M2 or M4; S-AML, secondaryAML; C-ALL, acute lymphoblastic leukemia, common antigen positive; T-ALL, ALL, T-cell phenotype; NR, no response;CR, complete response;NT, not tested. patients had de nova acute myeloid leukemia (AML), three patients had secondary AML (after Hodgkin’s disease: two patients, after myelodysplasia: one patient), three patients had acute lymphoblastic leukemia (ALL). Eight out of ten patients (nos l-8) were studied at presentation and two patients were studied at first or third relapse. Patients with AML were treated with an induction regimen consisting of daunorubicin given as an intravenous bolus dose of 45 mg/m’/day on days l-3 together with cytarabine, 200 mg/m’, given as a continuous intravenous infusion for 7 days. Second-line treatment consisted of amsacrine, 120 mg/m’/day intravenously on days 4-6 together with cytarabine, 1 g/m* intravenously, twice a day on days l-6. Patients with ALL were treated with an induction regimen consisting of daunorubicin given as an intravenous bolus dose of 4.5 mg/m’/day on days l-3 together with vincristine, 1.5 mg/m2/day intravenously, on days 1, 8, 15, 22, 29 and with asparaginase, 10.000 U/m2/day intravenously, on days 1,3,5,7,9,11, 13 and with prednisone, 60 mg/ m2/day orally, on days l-28. Second-line treatment consisted of amsacrine, 120 mg/m2/day intravenously, on days 1-5 together with cytarabine, 2 g/m2 intravenously, twice a day on days 4 and 5. Except one patient (no. lo), who was not treated any further, all patients failed induction treatment due to resistant leukemia, i.e. >30% blasts in the bone marrow without marrow hypoplasia, and all patients demonstrated blasts in the peripheral blood smears within 3 weeks of the induction treatment. Second-line treatment was given to eight out of nine patients, which resulted in complete remission in three out of eight patients. One patient did not receive second-line treatment because of clinical deterioration. Preparation of leukemic cells Bone marrow samples of untreated patients were collected in tubes containing preservative-free heparin.
Leukemic cells were obtained by Ficoll-Isopaque density gradient centrifugation, interphase mononuclear cells were recovered, and the cells were washed twice with phosphate-buffered saline and then resuspended in RPM1 1640 medium (Gibco Laboratories, Grand Island, NY, U.S.A.) containing 20% AB serum. Leukemic cells were purified further by T-cell depletion with sheep RBCS (in the case of T-cell leukemia, the separated Tcells were used after NH&l treatment). The samples always contained >95% blasts. The cells were resuspended in RPM1 1640 medium containing 20% AB serum and 10% dimethyl sulfoxide (Sigma Chemical Company, St. Louis, MO, U.S.A.) and cryopreserved until use by controlled-rate freezing in liquid nitrogen. For the experiments the cells were thawed rapidly, and the dimethyl sulfoxide concentration was lowered stepwise by dilution with cold RPM1 1640 medium containing 20% AB serum. Cells were washed at 4°C dead cells were removed by Ficoll-Isopaque density gradient centrifugation and the cells were diluted in RPM1 1640 medium containing 2% fetal bovine serum (Organon Teknika, Oss, The Netherlands) and 1% Lglutamine at a concentration of 5 x lo6 cells/ml. Viability of the cells was controlled by trypan blue dye exclusion. Treatment of the leukemic cells with ether lipids Purified blasts of the patients were placed in RPM1 1640 culture medium as described above in a 24-well plate (Nunc, Roskilde, Denmark), at a final volume of 1 ml/well and a final cell concentration of 5 x lo6 blasts/ml, and were incubated with medium (control), or with l-octadecyl-2-methyl-sn-glycerol-3-phosphocholine (ALP, Sigma) or with hexadecyl phosphocholine (Miltefosine, Asta Pharma AG, Frankfurt/Main, Germany) at a concentration of 10 &ml and of 50 l.@nl. Incubation times were 4 h for all experiments. AS a
Apoptosis of MDR+ leukemic cells by ether lipids
control, purified blasts were incubated simultaneously with cytarabine for 4 h at doses of 2 &ml and 5 lug/ml (corresponding to 10 -’ M and 2.5 x lo-’ M), and with daunorubicin for 2 h at a dose of 0.2 Kg/ml. Clonogenic assays and 3[H]thymidine incorporation for drug sensitivity Leukemic colony-forming cells (L-CFCs) could be generated from the blasts of all patients with AML. However, the blasts of the three patients with ALL failed to generate L-CFCs. In these instances, drug sensitivity was analyzed by proliferative assays with 3[H]thymidine incorporation. After drug exposure, the number of blasts was adjusted to achieve comparable cell concentrations for clonogenic growth assays. L-CFCs were cultured from the blasts in Iscove’s modified Dulbecco’s medium (IMDM, Gibco), containing 0.8% methylcellulose (Dow Chemical, Stade, Germany), 50 uM a-thioglycerol (Sigma), 18% AB serum, 100 U/ml rh GM-CSF (Boehringer, Mannheim, Germany) and 5 U/ml rh IL-3 (TNO, Rijswijk, The Netherlands). Aliquots of 1 ml containing 2 x lo5 cells/ml were cultured for 12-14 days in triplicate in 35 mm culture dishes (Greiner, Kunstoffwerke, Niirtingen, Germany) at 37% in 5% C02-humidified atmosphere and aggregates of >20 cells were enumerated as colonies. The numbers of colonies were calculated for each drug concentration tested. Cells in L-CFC were morphologically blasts. 3[H]thymidine incorporation in the blasts was analyzed by incubating the blasts in triplicate in microwells containing 5 x 10’ cells/well at 37°C in 5% C02humidified atmosphere for 16-20 h with 1 uCi of 3[H]thymidine (Radiochemical Centre, Amersham, U.K.), after which the cells were harvested. After harvesting, radioactivity incorporated into DNA was determined by liquid scintillation counting (cpm). Identically treated cells were irradiated (3000 rads) to give background values which were subtracted from each experiment. All studies dealing with drug exposure were performed for each patient at the same time and under identical circumstances. Clonogenic growth and “[Hlthymidine uptake experiments were performed in triplicate and the values obtained from these experiments are given as the mean for each single patient and are expressed as % of control values (medium only). Analysis of expression of MDRl Cellular expression of Pgp was determined by flow cytometry. The MDRl-specific monoclonal antibody MRK16 (Hoechst, Amsterdam, The Netherlands) was used. Aliquots containing 1 ml with 1 x lo6 purified blasts were washed twice in RPM1 1640 medium containing 1% bovine serum albumin and 0.5 x lo6 blasts were incubated with 10 pi MRK16 for 30 min at
39
4°C. Cells were washed and incubated with 20 pl FITCconjugated rabbit antimouse Ig antiserum (Dakopatts, Copenhagen, Denmark) for 30 min on ice, and subsequently were washed twice. Cells were analyzed on a FACScan (Becton Dickinson, Mountain View, CA, U.S.A.) and 5 000 events were counted. Control specimens using 0.5 x lo6 blasts and FITC-conjugated rabbit antimouse Ig antiserum, but without the specific monoclonal antibody, served as negative controls. The Pgp-positive cell line ZR-160 (small-cell lung cancer line; a generous gift from Dr R. J. Scheper, Department of Pathology, Free University Hospital, Amsterdam, The Netherlands) and the Pgp-negative cell line RPM1 8226 (plasma cell line; ATCC, Rockville, MD, U.S.A.) were used concurrently as positive and negative controls. An arbitrary limit of 3 30% blasts staining with one or both monoclonal antibodies, while negative in control staining, was considered positive. Previous studies have demonstrated that experiments with cryopreserved/ thawed blasts (if cryopreserved by controlled-rate freezing) give results similar to those performed with fresh blasts ([7], and personal observations). Rhodamine 123 eflux assay For Rh 123 efflux studies, viable purified blasts (only from patients with AML) were stained with 125 nmol/l Rh 123 for 10 min at 37°C and after two washes incubated in Rh 123-free medium containing the MDR inhibitor verapamil (50 umol/l). Cells were analyzed by flow cytometry. A threshold of 10% of cells showing Rh 123 efflux was defined as positive. Evaluation of apoptosis This was analyzed by binding of FITC-labeled annexin V (a generous gift from Dr C. P. M. Reutelingsperger, Department of Biochemistry, University of Limburg, Maastricht, The Netherlands) by flow cytometry, as described by Koopman et al. [32]. In brief, fresh purified blasts from two patients with acute leukemias (patient no. 10 and another patient with lymphoid transformation of chronic myeloid leukemia) were incubated in RPM1 1640 medium, supplemented with 2% fetal bovine serum and 1% L-glutamine, with FITC-labeled annexin V (final concentration of 2.5 ug/ ml) and, simultaneously, with miltefosine (at a concentration of 0, 10, 25, and 50 ug/ml) for 15, 45, 90, 120, and 240 min. Binding of FITC-labeled annexin V to the blasts after the various incubation times and concentrations with miltefosine was analyzed on a FACScan and 5 000 events were counted. This method detects apoptosis by the binding of (labeled) annexin V to the membrane phospholipid, phosphatidylserine, which is exposed on the outer leaflet of the cell membrane when cells enter into apoptosis.
L. F. Verdonck and H. G. van Heugten
40
Clonogenic Growth 100
Patient 1
1
1
90 I
Patient 4
Patient 3
Patient 2
1
80 -
0
ALP
70 _
[IIIIIIII Miltefosine
z g 0 z r; z cl a
60504030 _ 20 _ 10 -
Concentration (kg/ml)
Fig. 1. The cytotoxic effects of ALP and miltefosine on the leukemic colony growth of four patients with de nova AML who failed induction treatment. The blasts were exposed to 10 &ml and 50 &ml of both ether lipids for 4 h. Results are expressed as % of control leukemic cultures (without ether lipids).
Results
Incubation with ALP and miltefosine at 10 &ml and at 50 pg/ml for 4 h was studied. These variables were chosen because (a) Xl-100 pg ALP/ml for 4 h was generally used for purging studies [21,23] which demonstrated that these doses for 4 h induced a high cytotoxicity against L-CFCs with sparing of normal hematopoietic progenitor cells, and (b) miltefosine, given orally at 150 mg/day in a phase I/II study [25] for a median of 8 weeks, generated steady-state plasma levels of 40-80 @ml. Furthermore, low dosages of miltefosine (< 5 @ml) have a stimulating activity for normal hematopoietic progenitor cells and perhaps also for leukemic progenitors [33].
Cytotoxic effects of ether lipids All seven patients with primary refractory AML had L-CFCs, whereas these colonies could not be generated from the blasts of three patients with ALL. Cytotoxicity of ether lipids was analyzed by the elimination of LCFCs from the culture. The median number of L-CFCs observed with medium (control values) in these patients was 2015 (range 1004-8576) per 2 x lo5 blasts plated. Cytotoxicity of ether lipids was also quantitated by the inhibition of 3[H]thymidine incorporation (proliferative capacity) in the blasts of three patients with ALL. Clonogenic Growth Patlent 5
Patient 6
Patient 7
100 90 80
10
50
10
50
10
0
ALP
m
Miltefosine
50
Fig. 2. The cytotoxic effects of ALP and miltefosine on the leukemic colony growth of three patients with secondary AML who failed induction treatment. The blasts were exposed to 10 &ml and 50 @ml of both ether lipids for 4 h. Results are expressed as % of control leukemic cultures (without ether lipids).
41
Apoptosis of MDR+ leukemic cells by ether lipids ?H]Thymidine Incorporation Patient 10
Patient 9
Patient 8 100 7 90 -
E s 0 .& E 8 5 a
80 -
0
ALP
70 _
m
Miltefosine
60504030 _ 20 _ 10 -
10
50
IO
50
IO
50
Concentration (pg/ml)
Fig. 3. The cytotoxic effects of ALP and miltefosine on the ‘[Hlthymidine incorporation in leukemic blasts of three patients with ALL who failed induction treatment. The blasts were exposed to 10 ug/ml and 50 p&/ml of both ether lipids for 4 h. Results are expressed as % of control values (control ether lipids).
Incubation Time (m~nules) wth Annexin V 100 P d 2
go-
; t $ 4 8 P 8
70-
$
-
Patlent A
Patlent B
so-
60504030 _
25
50
10
0
25
50
pg Mllteloslne/ml mm 45 ml”
m
90 *in
m
120 mm
a
240 mm
Fig. 4. Binding of FITC-labeled annexin V to the blasts of two patients with MDRl-positive ALL after simultaneous exposure of the blasts to miltefosine at various drug levels and exposure times. Binding of FITC-labeled annexin V was analyzed by flow cytometry.
The results of 10 pg ALP/ml and 10 pg miltefosine/
ml are shown in Fig. 1. This dose of both ether lipids had some inhibiting effect against L-CFCs from patients with primary refractory de nova AML. However, 50 pg ALP/ml and 50 pg miltefosine/ml did eliminate all clonogenic blasts from these patients. Figure 2 shows the decrease in clonogenic growth of blasts from patients with secondary AML. The dose of 10 pg ALP/ml or 10 pg miltefosine/ml was barely cytotoxic against L-CFCs, whilst 50 pg/ml for both ether lipids was 100% effective. The inhibition of 3[H]thymidine incorporation by ether lipids in the blasts of three patients with ALL is shown in Fig. 3. Here too, 10 ps/ml of both ether lipids had some cytotoxic effects against L-
CFCs whilst 50 pg/ml of both ether lipids was very effective. Control experiments with cytarabine at doses of 2 pg/ ml and 5 @ml for 4 h demonstrated that a median of 11% (range O-39%) and 19% (range O-53%) respectively, of the L-CFCs were killed. Similar results were obtained with the proliferation assays using 3[H]thymidine incorporation. Control experiments with daunorubicin also showed that clonogenic AML cells were rather drug-resistant: a median of 32% (range O--93%) of L-CFCs were killed. Only one patient had daunorubicinsensitive L-CFCs (93% kill) which was in line with the low Pgp pump function of these blasts (12% Rh 123 efflux).
42
L. F. Verdonck and H. G. van Heugten
of MDRl Cellular expression of Pgp in purified blasts from the patients demonstrated that the blasts from all patients had a very high expression of Pgp: median 88% (range 82-100%) of the blasts were Pgp-positive. Efflux of Rh 123 showed comparable results: leukemic blasts of six out of seven patients with AML could be loaded with Rh 123 and blasts of five patients had Rh 123 efflux (>lO%). Median values were 59% (range 12-90%). AML blasts of only one patient did not have Rh 123 efflux (3%). In all positive cases, the efflux was blocked by verapamil.
Analysis
of apoptosis with binding of annexin V This was analyzed with fresh leukemic cells from two patients, before and after the in vitro treatment with miltefosine at various concentrations and various incubation times. As shown in Fig. 4, annexin V binding to the membranes of the blasts was 90-100% after incubation with 25 pg miltefosine/ml for only 15 min.
Detection
the fact that cytarabine and daunorubicin generally were unable to kill these clonogenic AML cells. Various mechanisms have been proposed to be of importance in the cytotoxic action of ether lipids [17, 18, 26-311 but most investigations point to direct damage of the cell membrane [l&26,28,31]. Recent investigations, however, suggest that ether lipids can also induce cell death by apoptosis [35,36]. Our results support the concept that ether lipids can kill acute leukemic blasts by the induction of apoptosis, which occurs very rapidly (within 15 min). In conclusion, ether lipids are able to induce apoptotic cell death in MDRl-positive leukemic cells, both from patients with drug-resistant AML and from patients with drug-resistant ALL. These anticancer drugs are not affected by the presence of the MDRl phenotype on leukemic cells. Clinical studies with this new class of anticancer drugs in patients with acute leukemias are warranted. References
Discussion This study suggests that ether lipids, a relatively new class of anticancer drugs, can be effective cytotoxic agents against MDRl-expressing acute leukemia cells. We have tested ex vivo two ether lipids, ALP and miltefosine, and both were equally effective against drug-resistant leukemic blasts of patients both with AML and with ALL. Like many other anticancer drugs, ether lipids can exert their cytotoxicity through the induction of apoptosis. To date, ether lipids are applied in the clinic as purging agents, because they are selectively toxic to leukemic cells [19,23], and as oral antitumor agents in phase I/II studies for patients with solid tumors [24,25,34]. These studies have demonstrated that leukemic blasts can effectively be killed with the ether lipid ALP at doses of 50 ug/ml for 4 h, whereas the normal hematopoietic stem cell is relatively spared and that with the oral agent miltefosine, at a daily dose of 3 x 50 mg, drug levels of 40-80 ug/ml can be achieved for prolonged times with acceptable side effects. Thus, it appears that ether lipid plasma levels of 50 @ml for at least 4 h can be achieved in vivo without unacceptable toxicity. In this study we tested ex vivo the cytotoxic effect of ether lipids against MDRl-positive leukemic blasts of patients who failed induction treatment due to resistant acute leukemias. Although 10 @ml for 4 h really was ineffective, 50 pg/ml for 4 h was 100% cytotoxic for the clonogenic leukemia cells in seven patients with primary refractory AML and also almost 100% cytotoxic for purified blasts of three patients with drug-resistant ALL. The efficacy of ether lipids was strengthened further by
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ETHER LIPIDS ARE EFFECTIVE CYTOTOXIC DRUGS AGAINST MULTIDRUG-RESISTANT ACUTE LEUKEMIA CELLS AND CAN ACT BY THE INDUCTION OF APOPTOSIS Leo F. Verdonck and Hans G. van Heugten Department of Haematology, University Hospital, Utrecht, The Netherlands (Received 19 June 1995. Revision accepted 20 April 1996) Abstract-We studied the cytotoxic effects of two ether lipids, a relatively new class of anticancer drugs, on multidrug-resistant (MDRI) leukemia cells of patients with acute leukemias who had failed induction treatment. The cytotoxicity of ether lipids was determined by the elimination of clonogenic leukemia cells from leukemic cultures or, in case of failure to generate leukemic cultures, by the inhibition of 3[Hlthymidine incorporation in leukemic blasts. At dose levels of 50 pg/ml, a plasma level that can be achieved after oral intake, the MDRIpositive blasts were killed, both of patients with drug-resistant acute myeloid leukemia (AML) and of patients with drug-resistant acute lymphoblastic leukemia (ALL). The leukemic blasts were killed by the induction of apoptosis. These data suggest that ether lipids may be effective antileukemic drugs and that their cytotoxic function is not affected by MDRI. 0 1997 Elsevier Science Ltd. Key words: Ether lipid, acute leukemia,
multidrug
resistance,
apoptosis.
[ll-161. Probably more attractive, however, will be the development of drugs that are not affected by MDRl. Previously, we and others have demonstrated that the ether lipid ET-18-OCH3 (ALP), a synthetic analog of 2lysophosphatidylcholine, exhibits selective antineoplastic activity to leukemic cells [ 17-201. This was followed by studies exploring the possibility of ALP (or its analogs) being an agent for purging bone marrow grafts of patients with acute leukemias for autologous marrow transplantation [19-231. Another ether lipid, hexadecylphosphocholine (miltefosine) has been investigated recently in clinical phase I/II studies as an oral anticancer drug for patients with solid tumors [24,25]. The mechanisms by which ether lipids exert their cytotoxic effects to malignant cells are under extensive investigation and several hypotheses have been proposed [17,18, 26-311. Here we report the results of in vitro studies showing that ALP and miltefosine effectively kill multidrugresistant blasts from patients with refractory acute leukemias and can act by the induction of apoptosis.
Introduction Although many patients with acute leukemias will achieve a complete remission with combination chemotherapy, the majority of patients eventually relapse because of the emergence or expansion of drug-resistant leukemic cells. Furthermore, some patients already have drug-resistant disease at presentation. Several mechanisms are recognized by which tumor cells become resistant to anticancer drugs [l]. Among these, the multidrug resistance gene (MDRl) encoding the transmembrane protein P-glycoprotein (Pgp), has been the subject of the majority of investigations [2-51. Pgp acts as a drug efflux pump, capable of expelling a variety of anticancer drugs from the cytosol [l-5]. Overexpression of MDRl results in an increased activity of Pgp and has been associated with drug resistance in patients with acute leukemias [6-lo]. This has led to the introduction of pharmacological interventions, e.g. the use of cyclosporine, which can competitively inhibit Pgp Abbreviations: ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; L-CFCs, leukemic colony-forming cells; MDRl, multidrug-resistant; Pgp, P-glycoprotein. Correspondence to: Leo F. Verdonck, MD, PhD, University Hospital Utrecht, Departmentof Haematology(G03.647),P.O. Box 85.500, 3508 GA Utrecht, The Netherlands (Tel: +31 30 50 72 30; Fax: +31 30 51 18 93; E-mail: [email protected]).
Materials and Methods Patients
Ten patients with acute leukemias were studied. The characteristics of the patients are given in Table 1. Four 37
38
L. F. VerdonckandH. G. van Heugten Table 1. Patient characteristics
Patient number 1 2 3 4 5 6 7 8 9 10
Sex/age
Diagnosis
M/39 M/33 M/32 Ml33 M/48 Ml46 M/32 M/25 F/23 F/44
AML, Ml AML, M4 AML, Ml AML, M2 S-AML, Ml S-AML, M2 S-AML, Ml C-ALL T-ALL (1st relapse) T-ALL (3rd relapse)
Responseto Induction treatment 2nd line treatment NR NR NR NR NR NR NR NR NR NT
CR CR NR NR NR NT CR NR NR NT
Abbreviations: AML, acute myeloblastic leukemia; FAH Classification Ml, M2 or M4; S-AML, secondaryAML; C-ALL, acute lymphoblastic leukemia, common antigen positive; T-ALL, ALL, T-cell phenotype; NR, no response;CR, complete response;NT, not tested. patients had de nova acute myeloid leukemia (AML), three patients had secondary AML (after Hodgkin’s disease: two patients, after myelodysplasia: one patient), three patients had acute lymphoblastic leukemia (ALL). Eight out of ten patients (nos l-8) were studied at presentation and two patients were studied at first or third relapse. Patients with AML were treated with an induction regimen consisting of daunorubicin given as an intravenous bolus dose of 45 mg/m’/day on days l-3 together with cytarabine, 200 mg/m’, given as a continuous intravenous infusion for 7 days. Second-line treatment consisted of amsacrine, 120 mg/m’/day intravenously on days 4-6 together with cytarabine, 1 g/m* intravenously, twice a day on days l-6. Patients with ALL were treated with an induction regimen consisting of daunorubicin given as an intravenous bolus dose of 4.5 mg/m’/day on days l-3 together with vincristine, 1.5 mg/m2/day intravenously, on days 1, 8, 15, 22, 29 and with asparaginase, 10.000 U/m2/day intravenously, on days 1,3,5,7,9,11, 13 and with prednisone, 60 mg/ m2/day orally, on days l-28. Second-line treatment consisted of amsacrine, 120 mg/m2/day intravenously, on days 1-5 together with cytarabine, 2 g/m2 intravenously, twice a day on days 4 and 5. Except one patient (no. lo), who was not treated any further, all patients failed induction treatment due to resistant leukemia, i.e. >30% blasts in the bone marrow without marrow hypoplasia, and all patients demonstrated blasts in the peripheral blood smears within 3 weeks of the induction treatment. Second-line treatment was given to eight out of nine patients, which resulted in complete remission in three out of eight patients. One patient did not receive second-line treatment because of clinical deterioration. Preparation of leukemic cells Bone marrow samples of untreated patients were collected in tubes containing preservative-free heparin.
Leukemic cells were obtained by Ficoll-Isopaque density gradient centrifugation, interphase mononuclear cells were recovered, and the cells were washed twice with phosphate-buffered saline and then resuspended in RPM1 1640 medium (Gibco Laboratories, Grand Island, NY, U.S.A.) containing 20% AB serum. Leukemic cells were purified further by T-cell depletion with sheep RBCS (in the case of T-cell leukemia, the separated Tcells were used after NH&l treatment). The samples always contained >95% blasts. The cells were resuspended in RPM1 1640 medium containing 20% AB serum and 10% dimethyl sulfoxide (Sigma Chemical Company, St. Louis, MO, U.S.A.) and cryopreserved until use by controlled-rate freezing in liquid nitrogen. For the experiments the cells were thawed rapidly, and the dimethyl sulfoxide concentration was lowered stepwise by dilution with cold RPM1 1640 medium containing 20% AB serum. Cells were washed at 4°C dead cells were removed by Ficoll-Isopaque density gradient centrifugation and the cells were diluted in RPM1 1640 medium containing 2% fetal bovine serum (Organon Teknika, Oss, The Netherlands) and 1% Lglutamine at a concentration of 5 x lo6 cells/ml. Viability of the cells was controlled by trypan blue dye exclusion. Treatment of the leukemic cells with ether lipids Purified blasts of the patients were placed in RPM1 1640 culture medium as described above in a 24-well plate (Nunc, Roskilde, Denmark), at a final volume of 1 ml/well and a final cell concentration of 5 x lo6 blasts/ml, and were incubated with medium (control), or with l-octadecyl-2-methyl-sn-glycerol-3-phosphocholine (ALP, Sigma) or with hexadecyl phosphocholine (Miltefosine, Asta Pharma AG, Frankfurt/Main, Germany) at a concentration of 10 &ml and of 50 l.@nl. Incubation times were 4 h for all experiments. AS a
Apoptosis of MDR+ leukemic cells by ether lipids
control, purified blasts were incubated simultaneously with cytarabine for 4 h at doses of 2 &ml and 5 lug/ml (corresponding to 10 -’ M and 2.5 x lo-’ M), and with daunorubicin for 2 h at a dose of 0.2 Kg/ml. Clonogenic assays and 3[H]thymidine incorporation for drug sensitivity Leukemic colony-forming cells (L-CFCs) could be generated from the blasts of all patients with AML. However, the blasts of the three patients with ALL failed to generate L-CFCs. In these instances, drug sensitivity was analyzed by proliferative assays with 3[H]thymidine incorporation. After drug exposure, the number of blasts was adjusted to achieve comparable cell concentrations for clonogenic growth assays. L-CFCs were cultured from the blasts in Iscove’s modified Dulbecco’s medium (IMDM, Gibco), containing 0.8% methylcellulose (Dow Chemical, Stade, Germany), 50 uM a-thioglycerol (Sigma), 18% AB serum, 100 U/ml rh GM-CSF (Boehringer, Mannheim, Germany) and 5 U/ml rh IL-3 (TNO, Rijswijk, The Netherlands). Aliquots of 1 ml containing 2 x lo5 cells/ml were cultured for 12-14 days in triplicate in 35 mm culture dishes (Greiner, Kunstoffwerke, Niirtingen, Germany) at 37% in 5% C02-humidified atmosphere and aggregates of >20 cells were enumerated as colonies. The numbers of colonies were calculated for each drug concentration tested. Cells in L-CFC were morphologically blasts. 3[H]thymidine incorporation in the blasts was analyzed by incubating the blasts in triplicate in microwells containing 5 x 10’ cells/well at 37°C in 5% C02humidified atmosphere for 16-20 h with 1 uCi of 3[H]thymidine (Radiochemical Centre, Amersham, U.K.), after which the cells were harvested. After harvesting, radioactivity incorporated into DNA was determined by liquid scintillation counting (cpm). Identically treated cells were irradiated (3000 rads) to give background values which were subtracted from each experiment. All studies dealing with drug exposure were performed for each patient at the same time and under identical circumstances. Clonogenic growth and “[Hlthymidine uptake experiments were performed in triplicate and the values obtained from these experiments are given as the mean for each single patient and are expressed as % of control values (medium only). Analysis of expression of MDRl Cellular expression of Pgp was determined by flow cytometry. The MDRl-specific monoclonal antibody MRK16 (Hoechst, Amsterdam, The Netherlands) was used. Aliquots containing 1 ml with 1 x lo6 purified blasts were washed twice in RPM1 1640 medium containing 1% bovine serum albumin and 0.5 x lo6 blasts were incubated with 10 pi MRK16 for 30 min at
39
4°C. Cells were washed and incubated with 20 pl FITCconjugated rabbit antimouse Ig antiserum (Dakopatts, Copenhagen, Denmark) for 30 min on ice, and subsequently were washed twice. Cells were analyzed on a FACScan (Becton Dickinson, Mountain View, CA, U.S.A.) and 5 000 events were counted. Control specimens using 0.5 x lo6 blasts and FITC-conjugated rabbit antimouse Ig antiserum, but without the specific monoclonal antibody, served as negative controls. The Pgp-positive cell line ZR-160 (small-cell lung cancer line; a generous gift from Dr R. J. Scheper, Department of Pathology, Free University Hospital, Amsterdam, The Netherlands) and the Pgp-negative cell line RPM1 8226 (plasma cell line; ATCC, Rockville, MD, U.S.A.) were used concurrently as positive and negative controls. An arbitrary limit of 3 30% blasts staining with one or both monoclonal antibodies, while negative in control staining, was considered positive. Previous studies have demonstrated that experiments with cryopreserved/ thawed blasts (if cryopreserved by controlled-rate freezing) give results similar to those performed with fresh blasts ([7], and personal observations). Rhodamine 123 eflux assay For Rh 123 efflux studies, viable purified blasts (only from patients with AML) were stained with 125 nmol/l Rh 123 for 10 min at 37°C and after two washes incubated in Rh 123-free medium containing the MDR inhibitor verapamil (50 umol/l). Cells were analyzed by flow cytometry. A threshold of 10% of cells showing Rh 123 efflux was defined as positive. Evaluation of apoptosis This was analyzed by binding of FITC-labeled annexin V (a generous gift from Dr C. P. M. Reutelingsperger, Department of Biochemistry, University of Limburg, Maastricht, The Netherlands) by flow cytometry, as described by Koopman et al. [32]. In brief, fresh purified blasts from two patients with acute leukemias (patient no. 10 and another patient with lymphoid transformation of chronic myeloid leukemia) were incubated in RPM1 1640 medium, supplemented with 2% fetal bovine serum and 1% L-glutamine, with FITC-labeled annexin V (final concentration of 2.5 ug/ ml) and, simultaneously, with miltefosine (at a concentration of 0, 10, 25, and 50 ug/ml) for 15, 45, 90, 120, and 240 min. Binding of FITC-labeled annexin V to the blasts after the various incubation times and concentrations with miltefosine was analyzed on a FACScan and 5 000 events were counted. This method detects apoptosis by the binding of (labeled) annexin V to the membrane phospholipid, phosphatidylserine, which is exposed on the outer leaflet of the cell membrane when cells enter into apoptosis.
L. F. Verdonck and H. G. van Heugten
40
Clonogenic Growth 100
Patient 1
1
1
90 I
Patient 4
Patient 3
Patient 2
1
80 -
0
ALP
70 _
[IIIIIIII Miltefosine
z g 0 z r; z cl a
60504030 _ 20 _ 10 -
Concentration (kg/ml)
Fig. 1. The cytotoxic effects of ALP and miltefosine on the leukemic colony growth of four patients with de nova AML who failed induction treatment. The blasts were exposed to 10 &ml and 50 &ml of both ether lipids for 4 h. Results are expressed as % of control leukemic cultures (without ether lipids).
Results
Incubation with ALP and miltefosine at 10 &ml and at 50 pg/ml for 4 h was studied. These variables were chosen because (a) Xl-100 pg ALP/ml for 4 h was generally used for purging studies [21,23] which demonstrated that these doses for 4 h induced a high cytotoxicity against L-CFCs with sparing of normal hematopoietic progenitor cells, and (b) miltefosine, given orally at 150 mg/day in a phase I/II study [25] for a median of 8 weeks, generated steady-state plasma levels of 40-80 @ml. Furthermore, low dosages of miltefosine (< 5 @ml) have a stimulating activity for normal hematopoietic progenitor cells and perhaps also for leukemic progenitors [33].
Cytotoxic effects of ether lipids All seven patients with primary refractory AML had L-CFCs, whereas these colonies could not be generated from the blasts of three patients with ALL. Cytotoxicity of ether lipids was analyzed by the elimination of LCFCs from the culture. The median number of L-CFCs observed with medium (control values) in these patients was 2015 (range 1004-8576) per 2 x lo5 blasts plated. Cytotoxicity of ether lipids was also quantitated by the inhibition of 3[H]thymidine incorporation (proliferative capacity) in the blasts of three patients with ALL. Clonogenic Growth Patlent 5
Patient 6
Patient 7
100 90 80
10
50
10
50
10
0
ALP
m
Miltefosine
50
Fig. 2. The cytotoxic effects of ALP and miltefosine on the leukemic colony growth of three patients with secondary AML who failed induction treatment. The blasts were exposed to 10 &ml and 50 @ml of both ether lipids for 4 h. Results are expressed as % of control leukemic cultures (without ether lipids).
41
Apoptosis of MDR+ leukemic cells by ether lipids ?H]Thymidine Incorporation Patient 10
Patient 9
Patient 8 100 7 90 -
E s 0 .& E 8 5 a
80 -
0
ALP
70 _
m
Miltefosine
60504030 _ 20 _ 10 -
10
50
IO
50
IO
50
Concentration (pg/ml)
Fig. 3. The cytotoxic effects of ALP and miltefosine on the ‘[Hlthymidine incorporation in leukemic blasts of three patients with ALL who failed induction treatment. The blasts were exposed to 10 ug/ml and 50 p&/ml of both ether lipids for 4 h. Results are expressed as % of control values (control ether lipids).
Incubation Time (m~nules) wth Annexin V 100 P d 2
go-
; t $ 4 8 P 8
70-
$
-
Patlent A
Patlent B
so-
60504030 _
25
50
10
0
25
50
pg Mllteloslne/ml mm 45 ml”
m
90 *in
m
120 mm
a
240 mm
Fig. 4. Binding of FITC-labeled annexin V to the blasts of two patients with MDRl-positive ALL after simultaneous exposure of the blasts to miltefosine at various drug levels and exposure times. Binding of FITC-labeled annexin V was analyzed by flow cytometry.
The results of 10 pg ALP/ml and 10 pg miltefosine/
ml are shown in Fig. 1. This dose of both ether lipids had some inhibiting effect against L-CFCs from patients with primary refractory de nova AML. However, 50 pg ALP/ml and 50 pg miltefosine/ml did eliminate all clonogenic blasts from these patients. Figure 2 shows the decrease in clonogenic growth of blasts from patients with secondary AML. The dose of 10 pg ALP/ml or 10 pg miltefosine/ml was barely cytotoxic against L-CFCs, whilst 50 pg/ml for both ether lipids was 100% effective. The inhibition of 3[H]thymidine incorporation by ether lipids in the blasts of three patients with ALL is shown in Fig. 3. Here too, 10 ps/ml of both ether lipids had some cytotoxic effects against L-
CFCs whilst 50 pg/ml of both ether lipids was very effective. Control experiments with cytarabine at doses of 2 pg/ ml and 5 @ml for 4 h demonstrated that a median of 11% (range O-39%) and 19% (range O-53%) respectively, of the L-CFCs were killed. Similar results were obtained with the proliferation assays using 3[H]thymidine incorporation. Control experiments with daunorubicin also showed that clonogenic AML cells were rather drug-resistant: a median of 32% (range O--93%) of L-CFCs were killed. Only one patient had daunorubicinsensitive L-CFCs (93% kill) which was in line with the low Pgp pump function of these blasts (12% Rh 123 efflux).
42
L. F. Verdonck and H. G. van Heugten
of MDRl Cellular expression of Pgp in purified blasts from the patients demonstrated that the blasts from all patients had a very high expression of Pgp: median 88% (range 82-100%) of the blasts were Pgp-positive. Efflux of Rh 123 showed comparable results: leukemic blasts of six out of seven patients with AML could be loaded with Rh 123 and blasts of five patients had Rh 123 efflux (>lO%). Median values were 59% (range 12-90%). AML blasts of only one patient did not have Rh 123 efflux (3%). In all positive cases, the efflux was blocked by verapamil.
Analysis
of apoptosis with binding of annexin V This was analyzed with fresh leukemic cells from two patients, before and after the in vitro treatment with miltefosine at various concentrations and various incubation times. As shown in Fig. 4, annexin V binding to the membranes of the blasts was 90-100% after incubation with 25 pg miltefosine/ml for only 15 min.
Detection
the fact that cytarabine and daunorubicin generally were unable to kill these clonogenic AML cells. Various mechanisms have been proposed to be of importance in the cytotoxic action of ether lipids [17, 18, 26-311 but most investigations point to direct damage of the cell membrane [l&26,28,31]. Recent investigations, however, suggest that ether lipids can also induce cell death by apoptosis [35,36]. Our results support the concept that ether lipids can kill acute leukemic blasts by the induction of apoptosis, which occurs very rapidly (within 15 min). In conclusion, ether lipids are able to induce apoptotic cell death in MDRl-positive leukemic cells, both from patients with drug-resistant AML and from patients with drug-resistant ALL. These anticancer drugs are not affected by the presence of the MDRl phenotype on leukemic cells. Clinical studies with this new class of anticancer drugs in patients with acute leukemias are warranted. References
Discussion This study suggests that ether lipids, a relatively new class of anticancer drugs, can be effective cytotoxic agents against MDRl-expressing acute leukemia cells. We have tested ex vivo two ether lipids, ALP and miltefosine, and both were equally effective against drug-resistant leukemic blasts of patients both with AML and with ALL. Like many other anticancer drugs, ether lipids can exert their cytotoxicity through the induction of apoptosis. To date, ether lipids are applied in the clinic as purging agents, because they are selectively toxic to leukemic cells [19,23], and as oral antitumor agents in phase I/II studies for patients with solid tumors [24,25,34]. These studies have demonstrated that leukemic blasts can effectively be killed with the ether lipid ALP at doses of 50 ug/ml for 4 h, whereas the normal hematopoietic stem cell is relatively spared and that with the oral agent miltefosine, at a daily dose of 3 x 50 mg, drug levels of 40-80 ug/ml can be achieved for prolonged times with acceptable side effects. Thus, it appears that ether lipid plasma levels of 50 @ml for at least 4 h can be achieved in vivo without unacceptable toxicity. In this study we tested ex vivo the cytotoxic effect of ether lipids against MDRl-positive leukemic blasts of patients who failed induction treatment due to resistant acute leukemias. Although 10 @ml for 4 h really was ineffective, 50 pg/ml for 4 h was 100% cytotoxic for the clonogenic leukemia cells in seven patients with primary refractory AML and also almost 100% cytotoxic for purified blasts of three patients with drug-resistant ALL. The efficacy of ether lipids was strengthened further by
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