Effects of homoharringtonine alone and in combination with ... - Nature

Leukemia (1997) 11, 624–628  1997 Stockton Press All rights reserved 0887-6924/97 $12.00

Effects of homoharringtonine alone and in combination with alpha interferon and cytosine arabinoside on ‘in vitro’ growth and induction of apoptosis in chronic myeloid leukemia and normal hematopoietic progenitors G Visani1, D Russo2, E Ottaviani1, P Tosi1, D Damiani2, A Michelutti2, S Manfroi1, M Baccarani2 and S Tura1 Institute of Hematology and Medical Oncology ‘Sera`gnoli’, University of Bologna; and 2Chair and Division of Hematology, Department of Medical and Morphological Research and Udine University Hospital, Udine, Italy

1

Homoharringtonine (HHT) is a cephalotaxine alkaloid that showed clinical efficacy in the chronic phase of chronic myeloid leukemia (Ph11CML). As a single agent, it resulted in effectively controlling leukocytosis and in producing sporadic karyotypic conversions; its clinical use in combination with interferon (IFN-a) for the treatment of CML could thus be considered. In this study we evaluated the growth inhibition and the induction of apoptosis determined by HHT alone and in combination with IFN-a and cytosine arabinoside (Ara-C) on normal and CML (both in chronic, CML-CP and in blastic phase; CML-BP) hematopoietic progenitors. HHT is able to determine a dose-dependent cell growth inhibition; evaluation of cytotoxic activity on semisolid cultures showed an activity significantly higher on CML-CP than on normal cells (P = 0.02 for HHT 50 ng/ml and P = 0.01 for HHT 200 ng/ml). HHT exerted a synergistic effect with IFN-a, Ara-C and IFN-a + Ara-C in inhibiting CML-CP colony growth; the same activity was demonstrated by the combination of HHT with Ara-C and by the triple combination, but not by HHT + IFN-a, on normal myeloid progenitors. The triple combination only was able to exert a synergistic effect in CML-BP. The induction of apoptosis resulted HHT dose-dependent in CML-CP and normals; at higher drug concentrations (100–200–1000 ng/ml), HHT induced a significant increase of apoptotic cells (for normals: P = 0.04, P = 0.02 and P = 0.04; for CML-CP: P = 0.01, P = 0.01 and P = 0.04, respectively); no significant changes were observed in CMLBP. In conclusion, the differences in cytotoxic effect and apoptosis induction observed, depending on the various phases of CML, add experimental evidence to the different clinical results between the chronic phase, where the clone is responsive to HHT, and the acute phase, where the drug is ineffective. The in vitro synergism of HHT with Ara-C and IFN-a in CML-CP suggests further evaluation in the clinical setting. Keywords: homoharringtonine; alpha interferon; cytosine arabinoside; chronic myeloid leukemia; cell cultures; apoptosis

be considered. New drug combinations including interferon (IFN-a) plus agents like Ara-C are being clinically tested in an attempt to improve the results of IFN-a alone.10 In fact, although IFN-a as a single agent induces a hematologic response in 60–80% of patients, less than 30–40% of the responding patients obtain both a karyotypic conversion, varying from minor to complete (Ph negative metaphases from 33 to 100%), and a significant increase of the duration of the chronic phase.11–15 Based on these premises, we evaluated the in vitro growth inhibition and the induction of apoptosis of HHT alone and in combination with IFN-a and Ara-C on normal and CML (both in chronic and in blastic phase) hematopoietic progenitors. Materials and methods

Reagents HHT was purchased from Pharmacia, Chang Zhou, China. Ara-C was purchased from Sigma Chemicals (St Louis, MO, USA); IFNa (3 × 106 U/mg) was kindly provided by HoffmanLa Roche (Basel, Switzerland). Drugs were initially dissolved in normal saline; further dilutions were performed in Iscove’s modified Dulbecco’s medium (IMDM; Gibco Europe, Paisley, UK). The doses of IFN-a, Ara-C and HHT were chosen on the basis of dose–response curves and previously reported data.6,8,16,17

Bone marrow cell samples Introduction Homoharringtonine (HHT), is a cephalotaxine alkaloid1 shown to have a clinical efficacy in the treatment of de novo acute myeloid leukemia (AML) either alone or in combination with other drugs, mainly cytosine arabinoside (Ara-C).2–5 The inhibition of protein synthesis was identified as the most important mechanism of its antileukemic activity.6,7 However, the induction of apoptosis was further demonstrated in the chronic phase of chronic myeloid leukemia (Ph1+CML) in which HHT, as a single agent, resulted in being also effective in controlling leukocytosis and in producing a sporadic karyotypic conversion.8,9 Because of the activity of HHT on the CML clone its clinical use in combination with IFN-a for the treatment of CML could

Correspondence: G Visani, Istituto di Ematologia e Oncologia Medica ‘Sera`gnoli’, Policlinico S, Orsola, Via Massarenti, 9, 40138 Bologna, Italy Received 13 October 1995; accepted 30 December 1996

Heparinized bone marrow samples from 10 normals (five males and five females, mean age 37 years), 10 CML chronic phase (CML-CP) (six males and four females, mean age 39 years) and 10 CML blastic phase (CML-BP) (four males and six females, mean age 41 years) patients were studied after informed consent. CML-CP patients were studied at diagnosis, before any therapy. They received, during the chronic phase, a standard treatment with hydroxyurea, according to white cell count, platelet count, and spleen enlargement, as previously described.11 Mononuclear cells were collected after sedimentation on Ficoll–Hypaque (Lymphoprep; Nycomed Pharma, Oslo, Norway) T depleted by E-rosetting and deprived of adherent cells, as previously described.18 The final percentage of T cells was less than 1% in each sample. After two washings cells were resuspended in IMDM.

Short-term cell cultures Cells (at a final concentration of 3 × 105/ml) were plated in 1 ml IMDM supplemented with 6.7% FCS, 6.7% horse serum

Homoharringtonine, IFN-a and Ara-C in chronic myeloid leukemia G Visani et al

(Gibco Europe, 6.7% trypticase soy broth (Becton Dickinson, Cockeysville, MD, USA), 6.7% dyalised bovine serum albumin (BSA) (Sigma Chemicals), 3 × 10−5 M egg lecithin (Merck, Darmstadt, Germany), 10−7 M sodium selenite (Merck), 7.7 × 10−6 M iron saturated human transferrin (Behring Institute, Marburg, Germany) and 10−4 M 2-mercaptoethanol. HHT alone was added at the following concentrations of 10, 50, 200 ng/ml. HHT at the concentration of 50 ng/ml in combination with Ara-C (100 ng/ml) and/or IFN-a (1000 U/ml) was further tested. The final suspension was seeded into 24-well plates; after 72 h incubation cells were resuspended and counted, as previously described.17,19,20 Parallel experiments, with analysis, after 7 days of the colony formation (aggregates composed of more than 50 cells) were performed.

Table 1 Cell growth inhibition (in comparison to control) due to different concentrations of HHT

HHT concentration (ng/ml) 10 50 200

CML-CP

CML-BP

Normals

28.8 ± 6.9 35.7 ± 7.1 54.7 ± 6.2

22.0 ± 5.4 33.0 ± 2.0 47.3 ± 2.6

13.9 ± 5.7 31.4 ± 5.1 42.4 ± 6.5

Values are expressed as m ± s.e.

Evaluation of drug combinations: mean fractional product The activity of the drug combinations was calculated by the mean fractional product as previously reported.21–23 Briefly, the ratio between the product of the survival fraction for each agent alone (expected survival) and the survival fraction observed with the drug combination was calculated. Values greater than 1 were considered to show synergism; those less than 0.9–1, ineffective and those of about 1, an additive effect.

Semisolid cultures CFU-GM colonies were cultured as previously described.18,24,25 Briefly, 1 × 105 cells were plated in 1 ml IMDM containing 0.9% methylcellulose, 20% FCS and 10% phytohemoagglutinin-stimulated lymphocyte-conditioned medium (PHA-LCM) as a source of colony stimulating activity. HHT was added at the following concentrations (ng/ml): 0.1, 1,10, alone and in combination (1 ng/ml) with Ara-C (1 ng/ml) and/or IFN-a (100 U/ml). Cells were plated in Petri dishes (35 × 10 mm) and CFU-GM colonies (aggregates composed of more than 50 cells) were counted after 14 days of culture at 37°C in a fully humidified atmosphere of 5% CO2 in air. Growth inhibition was calculated as a percentage of control (no drug in the sample) colony growth.

Evaluation of apoptosis Apoptosis was quantitated by flow cytometry, as previously described.26 Cells (2.5 × 106) were incubated in 10 ml IMDM plus 10% heat-inactivated fetal calf serum (FCS; Gibco). Samples were incubated for 24 h with various concentrations of HHT (ng/ml: 10, 100, 200, 1000). Control samples received the same amount of media, without drug addition. After 24 h of incubation the samples were pelleted and fixed in ethanol 70% for 15 min at 4°C; after three washes in PBS, the cells were treated with RNase I (Sigma) 0.5 mg/ml for 15 min at 37°C. The cells were harvested by centrifugation and resuspended in 50 mg/ml propidium iodide (Sigma) in PBS. Analysis (upon acquisition of 10 000–20 000 events) was performed on a FACscan flow cytometer (Becton Dickinson, San Jose, CA, USA) with the FL2 detector in logarithmic mode, using Lysis II software (Becton Dickinson). Apoptotic cells were located in the hypodiploid region of the histogram, due to chromosome condensation and fragmentation.

Figure 1 Cytotoxic effect of different concentrations of HHT (10,50,200 ng/ml) on normal and CML (both in chronic and in blastic phase) hematopoietic progenitors. Cytotoxic effect is evaluated as growth inhibition in comparison to controls.

Statistical analysis Data were analyzed by Student’s t-test. P values were considered significant when ,0.05. Results

Cytotoxic effect of HHT The cytotoxic effect of HHT is shown in Table 1 and Figure 1. In particular, at different concentrations of HHT (10, 50, 200 ng/ml) the cell growth inhibition on CML-CP was 28.8 ± 6.9, 35.7 ± 7.1 and 54.7 ± 6.2, respectively. Evaluation of HHT cytotoxic activity on semisolid cultures in shown in Table 2. The IC50 of HHT in CML-CP and in normal bone marrow cells was 0.6 ng/ml and 1.7 ng/ml, respectTable 2 CFU-GM growth inhibition (in comparison to controls) due to different concentrations of HHT

HHT concentration (ng/ml) 0.1 1 10

CML-CP

Normals

P value

64.6 ± 5.3 45.6 ± 5.9 31.4 ± 5.6

69.6 ± 5.3 56.0 ± 5.8 40.2 ± 3.0

NS 0.02 0.01

Values are expressed as m ± s.e. NS, not significant.

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ively. HHT showed a significantly higher cytotoxic activity on CML-CP (HHT 1 ng/ml: P = 0.02; HHT 10 ng/ml: P = 0.01).

Table 4 Evaluation of drug combinations on normal hematopoietic progenitors

% of cell growth

Cytotoxic effects of combinations Short-term cultures: The effects of the various drug combinations (HHT (50 ng/ml) and/or IFN-a and/or Ara-C) on CML (CP and BP) and on normal progenitor cells, evaluated in short-term cultures and calculated by the mean fractional product, are reported in Tables 3–5. HHT exerts a synergistic effect with IFN-a, Ara-C and IFN-a + Ara-C in inhibiting CMLCP colony growth (Table 3A); the same activity was demonstrated by the combination of HHT with Ara-C and by the triple combination but not by HHT + IFN-a on normal myeloid progenitors (Table 4A). In CML-BP only the triple combination was able to exert a synergistic effect (Table 5A). HHT exerted an additive effect with IFN-a and Ara-C in inhibiting the cell growth of CML-CP, CML-BP and normal cells (MFP respectively of 1.0 and 0.9 for CML-CP; 0.9 and 1.1 for CML-BP; 1.0 and 0.9 for normals).

Semisolid cultures: HHT exerted an additive effect with Ara-C (MFP = 1.0); the triple combination was able to exert an additive/synergistic effect on CML-CP progenitors (MFP = 1.2) (Table 3C). In normal cell progenitors the triple combination exerted an additive effect (MFP = 1.0) while the combination of HHT with Ara-C was able to exert an additive/synergistic effect (MFP = 1.2) (Table 4C).

(A) Short-term cultures: colony growth IFN-a 74.8 ± 7.4 Ara-C 71.3 ± 5.2 HHT 50 ng/ml 14.9 ± 5.8 IFN-a + HHT 13.6 ± 7.4 Ara-C + HHT 7.9 ± 5.1 Ara-C + IFN-a 37.7 ± 8.9 Ara-C + IFN-a + HHT 2.0 ± 1.6

0.8 1.3 synergistic 1.4 synergistic 2.8 synergistic

(B) Short-term cultures: cell growth IFN-a 88.4 ± 6.2 Ara-C 74.5 ± 4.8 HHT 50 ng/ml 68.6 ± 5.1 IFN-a + HHT 60.9 ± 7.1 Ara-C + HHT 58.7 ± 4.4 Ara-C + IFN-a 57.5 ± 4.0 Ara-C + IFN-a + HHT 62.4 ± 6.8

1.0 additive 0.9 additive 1.1 additive 0.6

(C) Semisolid cultures: CFU-GM IFN-a 65.6 ± 4.9 Ara-C 51.0 ± 6.6 HHT 1 ng/ml 56.0 ± 5.8 38.8 ± 3.1 IFN-a + HHT Ara-C + HHT 24.6 ± 7.2 Ara-C + IFN-a Ara-C + IFN-a + HHT

43.6 ± 8.2 23.4 ± 7.2

% of cell growth

(A) Short-term cultures: colony growth 81.0 ± 5.7 IFN-a Ara-C 69.7 ± 13.9 HHT 50 ng/ml 29.7 ± 8.4 IFN-a + HHT 12.5 ± 1.5 Ara-C + HHT 3.2 ± 3.2 Ara-C + IFN a 18.2 ± 4.4 1.0 ± 1.0 Ara-C + IFN-a + HHT (B) Short-term cultures: cell growth 79.2 ± 10.1 IFN-a Ara-C 72.8 ± 7.8 HHT 50 ng/ml 64.3 ± 7.1 IFN-a + HHT 51.8 ± 3.4 Ara-C + HHT 50.3 ± 8.1 59.8 ± 4.2 Ara-C + IFN-a 51.5 ± 6.9 Ara-C + IFN-a + HHT (C) Semisolid cultures: CFU-GM IFN-a 51.4 ± 7.0 Ara-C 81.2 ± 6.9 HHT 1 ng/ml 45.6 ± 5.9 IFN-a + HHT 25.0 ± 2.8 Ara-C + HHT 35.6 ± 5.4 Ara-C + IFN-a 37.2 ± 4.6 14.6 ± 2.8 Ara-C + IFN-a + HHT

Mean fractional product

1.9 6.4 3.1 5.3

synergistic synergistic synergistic synergistic


0.9 additive 1.0 additive 1.1 additive 1.2 additive/synergistic

0.9 additive 1.2 additive/synergistic 0.8 1.0 additive

Table 5 Evaluation of drug combinations on LMC-BP hematopoietic progenitors

% of cell growth Table 3 Evaluation of drug combinations on CML-CP hematopoietic progenitors


(A) Short-term cultures: colony growth IFN-a 64.2 ± 6.0 Ara-C 71.4 ± 15.3 HHT 50 ng/ml 30.8 ± 8.4 IFN-a + HHT 19.4 ± 12.0 Ara-C + HHT 24.4 ± 7.9 Ara-C + IFN-a 46.2 ± 10.1 Ara-C + IFN-a + HHT 11.6 ± 6.3 (B) Short-term cultures: cell growth 79.6 ± 4.1 IFN-a Ara-C 78.8 ± 2.3 HHT 50 ng/ml 67.0 ± 1.6 62.2 ± 7.8 IFN-a + HHT Ara-C + HHT 48.4 ± 2.5 Ara-C + IFN-a 55.2 ± 4.2 59.0 ± 7.2 Ara-C + IFN-a + HHT


1.0 additive 0.9 additive 1.0 additive 1.2 additive/ synergistic


Evaluation of apoptosis induction The induction of apoptosis resulted HHT dose-dependent in CML-CP and normal cells; at the higher tested drug concentrations (100, 200, 1000 ng/ml), HHT induced a significant increase of apoptotic cells (respectively P = 0.04, P = 0.02 and P = 0.04 for normals; P = 0.01, P = 0.01 and P = 0.04 for CMLCP). In addition, the increase of apoptotic cells induced by HHT 10 ng/ml in CML-CP was significant (if compared to control) (P = 0.05). In CML-BP no significant changes were


detected (a slight increase only at the higher drug concentration) (Table 6). Discussion Karyotypic remission is the major goal of therapy in CML, as it has been demonstrated that survival is directly related to the degree of karyotypic conversion.11 However, a large proportion of patients treated with IFN-a still do not achieve a karyotypic conversion or obtain a minimal karyotypic response (Ph-negative metaphases ,33%). For this reason, new effective drug combinations including IFN-a are clinically tested, in an attempt to increase the percentage of karyotypic responses. Promising clinical results seem to come from the association of IFN-a + Ara-C, but there is no conclusive evidence suggesting an advantage from this combination.10,11 Based on its activity on the CML clone, HHT could be considered as an interesting drug for clinical tests in combination with IFN-a.9,27 On this basis, we studied the in vitro effects of HHT on CML cells, both alone and in combination with IFNa and/or Ara-C. HHT was able to inhibit cell growth in CMLCP, CML-BP and normal cells in a dose-dependent way. HHT (1–10 ng/ml) was able to inhibit significantly CFU-GM growth of CML-CP vs normal cells (P = 0.02 and P = 0.01, respectively). Apoptosis data were in agreement with growth inhibition experiments of CML-CP and normals, as its induction resulted dose dependent, whereas no effect was seen in CML-BP. These data are in line with recent findings showing that HHT belongs to the category of MDR-related drugs, its antileukemic effect being modulated by P170 glycoprotein.27,28 In fact, P170 expression is increased in CML-BP in comparison to CML-CP;2,5,29,30 the significantly different cytotoxicity and apoptosis induction observed by us provided experimental evidence for the different clinical results observed in chronic phase, where HHT is effective, and in acute phase, where the drug is ineffective. Furthermore, an increased P170 expression has been demonstrated in early normal progenitors (CD34+) in comparison to the more differentiated (committed) compartment. Thus, the increased cytotoxicity both of HHT and of the combination HHT + IFN-a in CML chronic phase in comparison to the normal could be explained by an increased expression of the ‘committed progenitors’ pool. However, a possible higher senTable 6 Induction of apoptosis in normal bone marrow cells, CML chronic phase and CML acute phase cells using different concentrations of HHT (10,100,200,1000 ng/ml)

CTR (no drugs) HHT 10 ng/ml HHT 100 ng/ml HHT 200 ng/ml HHT 1000 ng/ml

Normals

CML-CP

CML-BP

1.34 ± 0.98%

23.8 ± 16.6%

22.0 ± 7.9%

3.3 ± 1.8% NS 5.6 ± 2.5% P = 0.04 10.1 ± 3.6% P = 0.02 13.4 ± 5.6% P = 0.04

25.7 ± 16.4% P = 0.05 30.4 ± 17.8% P = 0.01 37.9 ± 20.6% P = 0.01 50.4 ± 19.3% P = 0.04

23.9 ± 14.5% NS 26.7 ± 4.6% NS 21.6 ± 10.3 NS 31.7 ± 8.6 NS

CTR, controls; HHT, homoharringtonine; CML-CP, chronic myeloid leukemia-chronic phase; CML-BP, chronic myeloid leukemia-blastic phase; NS, not significant.

sitivity of normal marrow progenitors should be considered; studies on purified staminal populations could help to explain these points and are currently under evaluation in our laboratory. As an alternative, interactions of IFN-a on the posttranscriptional modification of P170 should also be considered.31,32 The in vitro synergisms of HHT with Ara-C and/or IFN-a on CML-CP cells add further evidence of a selective effect both of HHT and of the combinations HHT + IFN-a and HHT + AraC, in chronic myeloid leukemia in chronic phase, supporting further evaluation in the clinical setting.

Acknowledgements This work was supported in part by MURST 40%–60%.

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