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Targeting of heat shock protein 32 (Hsp32)/heme oxygenase-1 (HO-1) in leukemic cells in chronic myeloid leukemia: a novel approach to overcome resistance against imatinib

Matthias Mayerhofer, Karoline V. Gleixner, Julia Mayerhofer, Gregor Hoermann, Eva Jaeger, Karl J. Aichberger, Rene G. Ott, Khaled Greish, Hideaki Nakamura, Sophia Derdak, Puchit Samorapoompichit, Winfried F. Pickl, Veronika Sexl, Harald Esterbauer, Ilse Schwarzinger, Christian Sillaber, Hiroshi Maeda and Peter Valent

Article Figures & Data

Figures

  • Figure 1

    Knockdown of expression of HO-1 in CML cells is followed by apoptosis. (A) K562 cells were left untreated (Control) or were transfected with a control siRNA against luciferase or an siRNA specific for HO-1 as indicated. 8 hours after transfection, the percentage of apoptotic cells (provided for each condition) was determined by combined annexin V/propidium iodide staining and FACS analysis. PI, propidium iodide. (B) K562 cells were incubated in control medium (Co) or were transfected with control siRNA against Luciferase (Luc) or with a HO-1-specific siRNA (HO-1) at 100 nM or 200 nM. 8 hours after transfection, expression of HO-1 was determined by Western blotting. Expression of β-actin is shown as loading control. (C) The effects are shown for 3 different siRNAs (nos. 1–3) specific for HO-1 on viability of K562 cells compared with untransfected cells (Co) or cells transfected with a control siRNA (Luc). Results represent the mean (± SD) of 3 independent experiments. *P < .05 compared with control (Co).

  • Figure 2

    Pharmacologic inhibition of HO-1 suppresses growth of CML cell. (A,B) K562 cells were treated with PEG-ZnPP (left panel) or SMA-ZnPP (right panel) (5 μM each) for various time periods as indicated. Thereafter, cellular uptake of the compounds expressed as μM ZnPP incorporated in 106 input cells (A), and the HO-1 activity in treated cells (B) were determined as described in “Methods.” HO-1 activity is expressed as nanomoles of bilirubin formed per milligram of protein per hour (nmol Bilirubin/mg/hr). Results represent the mean (± SD) of 3 independent experiments. *P < .05 compared with untreated cells (timepoint 0). (C) Time-dependent inhibition of growth of K562 cells by SMA-ZnPP. K562 cells were incubated with SMA-ZnPP (10 μM; gray bars) or control medium (black bars) at 37°C for various time periods as indicated. Proliferation was measured by 3H-thymidine incorporation. Results are expressed as percent of medium control and represent the mean (± SD) of triplicates of one representative experiment. (D-G) K562 cells (D,E) or KU812 cells (F, G) were incubated with various concentrations of PEG-ZnPP (D,F) or SMA-ZnPP (E, G) as indicated for 48 hours. After incubation, growth of cells was determined by 3H-thymidine incorporation. Results represent the mean (± SD) of 3 independent experiments. *P < .05 compared with control.

  • Figure 3

    PEG-ZnPP and SMA-ZnPP inhibit growth of primary CML cells. Isolated leukemic cells from patients with newly diagnosed CP CML (A,B) or advanced phase CML (C: AP; D: BP) were stimulated with GM-CSF (100 U/mL) for 7 days and then incubated with control medium (0), various concentrations of PEG-ZnPP (left panels), or various concentrations of SMA ZnPP (right panels) at 37°C for 48 hours. Thereafter, 3H-thymidine incorporation was determined. Results show the percentage of 3H-thymidine uptake compared with control (100%) and represent the mean (± SD) of triplicates in each patient. (E) Sorted pure CD34-positive progenitor cells from a patient with newly diagnosed CP CML were incubated with control medium (0) or various concentrations of SMA-ZnPP at 37°C for 48 hours. Thereafter, 3H-thymidine uptake was determined. Results are expressed as percent 3H-thymidine uptake compared with control (100%) and represent the mean (± SD) of triplicates. (F) CD34-positive progenitor cells from 3 CML patients were incubated with control medium (Co) or SMA-ZnPP (20 μM) for 48 hours. Thereafter, 3H-thymidine incorporation was determined. Results represent the mean (± SD) of 3 independent experiments (3 patients). *P < .05 compared with control. (G) Isolated CD34+ CML progenitor cells were cultured in control medium (left panel) or in the presence of SMA-ZnPP (10 μM) (right panel) for 4 days. Thereafter, the percentage of apoptotic cells was determined by annexin V staining and FACS analysis.

  • Figure 4

    Hsp32/HO-1-targeting drugs inhibit growth of imatinib-resistant CML cells. (A) Imatinib-resistant K562 cells were incubated in medium containing various concentrations of ZnDPPIX (left), PEG-ZnPP (middle), or SMA-ZnPP (right) for 48 hours. Growth-inhibitory effects were quantified by 3H-thymidine incorporation assay. Results show the percentage of 3H-thymidine uptake compared with medium control (100%) and represent the mean (± SD) of 3 independent experiments. *P < .05 compared with medium control. (B) Ba/F3 cells expressing wt BCR/ABL or various imatinib-resistant mutants of BCR/ABL were incubated with various concentrations of SMA-ZnPP for 48 hours. Thereafter, proliferation was measured by 3H-thymidine incorporation. Results are expressed as percent of control and represent the mean (± SD) of triplicates from one representative experiment. (C) Isolated leukemic cells from 3 patients with imatinib-resistant CML were treated with PEG-ZnPP for 48 hours. Thereafter, proliferation was measured by 3H-thymidine incorporation assay. Results are expressed as percent of control and represent the mean (± SD) of 3 independent experiments (3 patients). *P < .05 compared with control. (D) Isolated leukemic cells from a patient with imatinib-resistant CML carrying BCR/ABL T315I, were treated with PEG-ZnPP (left panel) or SMA-ZnPP (right panel) for 48 hours. Thereafter, 3H-thymidine incorporation was measured. Results show the percentage of 3H-thymidine uptake compared with control (100%) and represent the mean (± SD) of triplicates. (E) Ba/F3p210wt (left panel) and Ba/F3p210T315I cells (right panel) were injected subcutaneously into nude mice (4 in each group). 7 days after tumor inoculation, when visible tumors had developed, mice were treated with PEG-ZnPP (3 mM, 0.1 mL, equivalent to 5 mg of ZnPP/kg) intravenously 3 times a week over a period of 2 weeks. Mice in the control group received physiological saline instead of PEG-ZnPP. On day 30, mice were killed, and tumor nodules were excised and weighed.

  • Figure 5

    SMA-ZnPP induces apoptosis in CML cells. (A) K562 cells were incubated with control medium (left panel) or SMA-ZnPP (10 μM) (right panel) for 48 hours. Thereafter, cells were analyzed by electron microscopy as described in “Methods.” SMA-ZnPP was found to induce typical morphological signs of apoptosis (chromatin fragmentation/condensation). (B) K562 cells were incubated with control medium (left panel) or SMA-ZnPP (10 μM) (right panel) for 48 hours. Thereafter, cells were subjected to a Tunel assay. Apoptotic cells display bright green fluorescence. (C) K562 cells were incubated in control medium (Co) or in PEG-ZnPP or SMA-ZnPP (each 10 or 20 μM) at 37°C. After 48 hours, cells were subjected to annexin-V staining. Results show the percentage of annexin-V–positive cells and represent the mean ± SD of 3 independent experiments. *P < .05 compared with control. (D) K562-KRAB-HO-1 cells were cultured in control medium (Co) or in the presence of doxycycline (1 μg/ml) for 24 hours. Thereafter, expression of Hsp32/HO-1 was determined by Western blotting. β-actin is shown as loading control. (E) K562-KRAB-HO-1 cells were cultured in control medium (Co) or in the presence of doxycycline (1 μg/mL) for 24 hours. Thereafter, cells were kept in the absence or presence of SMA-ZnPP (10 μM) as indicated for 48 hours. Cell viability was determined by trypan blue exclusion test. Results show the percentage of trypan blue-positive cells and represent the mean (± SD) of 3 independent experiments. (F) Leukemic cells from untreated patients with CP CML were cultured in control medium or in the presence of SMA-ZnPP (10 μM or 30 μM) for 4 days. Thereafter, the percentage of apoptotic cells was determined by annexin-V staining and FACS analysis. Results represent the mean (± SD) of 3 patients. *P < .05. (G) Primary CML cells were cultured in the absence or presence of SMA-ZnPP (10 μM) for 5 days. Thereafter, the percentage of apoptotic cells was quantified on Wright-Giemsa-stained cytospin preparations. Results represent the mean (± SD) of 3 patients. *P < .05.

  • Figure 6

    HO-1-targeting drugs synergize with BCR/ABL tyrosine kinase. (A) Imatinib-resistant K562 cells were grown in the absence of imatinib for 24 hours, and then were incubated with various concentrations of imatinib (■-■), ZnDPPIX (•-•), or a combination of imatinib and ZnDPPIX at a constant ratio of 1:200 (▴-▴) at 37°C for 48 hours. Thereafter, proliferation was measured by 3H-thymidine incorporation assay. Results are expressed as percent of medium control and represent the mean (± SD) of triplicates. (B) Combination index (CI) values were determined for each fraction affected as described in “Methods.” A CI value of 1.0 indicates an additive effect, a CI greater than 1.0 indicates antagonism, whereas a CI less than 1.0 indicates synergism. (C) Ba/F3p210T315I cells were incubated with various concentrations of imatinib (■-■), SMA-ZnPP (•-•), or a combination of imatinib and SMA-ZnPP at a constant ratio of 1:0.1 (▴-▴). (D) Ba/F3p210T315I cells were incubated with various concentrations of nilotinib (■-■), SMA-ZnPP (•-•), or a combination of nilotinib and SMA-ZnPP at a constant ratio of 1:0.4 (▴-▴). After incubation at 37°C for 48 hours, cell growth was determined by 3H-thymidine incorporation. Results are expressed as percent of medium control and represent the mean (± SD) of triplicates of one representative experiment for each drug combination. (E) Ba/F3p210T315I cells were cultured in control medium, imatinib (10 μM), PEG-ZnPP (10 μM), SMA-ZnPP (1 μM), or combinations of drugs as indicated, for 48 hours. Thereafter, cell viability was determined by trypan blue exclusion test. Results show the percentage of viable (trypan blue-negative) cells and represent the mean (± SD) of 3 independent experiments. (F) Primary leukemic cells from a patient carrying the BCR/ABL T315I mutation were cultured in control medium, PEG-ZnPP (5 μM), imatinib (5 μM), or a combination of both compounds (1:1 ratio) for 48 hours. Thereafter, proliferation was determined by 3H-thymidine incorporation. Results are expressed as percent of medium control and represent the mean (± SD) of triplicates. (G) Primary leukemic cells from a patient carrying BCR/ABL T315I were cultured in control medium, SMA-ZnPP (7.5 μM), imatinib (0.75 μM), or a combination of both compounds (10:1 ratio) for 48 hours. Thereafter, proliferation was determined by 3H-thymidine incorporation. Results are expressed as percent of medium control and represent the mean (± SD) of triplicates. (H) PB MNC from 3 healthy donors were cultured in control medium, imatinib (10 μM), PEG-ZnPP (10 μM), SMA-ZnPP (1 μM), or combinations of these drugs as indicated for 48 hours. Thereafter, cell viability was determined by trypan blue exclusion test. Results show the percentage of viable (trypan blue–negative) cells and represent the mean (± SD) of 3 independent experiments (3 donors).

Tables

  • Table 1

    Patients' characteristics and response to Hsp32/HO-1-targeted drugs

    Patient no.SexAge, yCML phaseImatinib-resistantWBC, G/LHb g/dLPlt, G/LCytogeneticsBCR/ABL point mutationsIC50 SMA-ZnPPIC50 PEG-ZnPP
    1M47BPYes30.410.6141t(9;22), i17, +8Glu279/Lys His396/Arg5n.t.
    2F46CPNo38.811.9569t(9;22)wt10n.t.
    3M49BPNo16.5812.2871t(9;22)wt15n.t.
    4M59CPNo159.412.4168t(9;22)n.t.3n.t.
    5F80CPNo17.113.7247t(9;22)n.t.n.t.2
    6M67APYes49.514.1638t(9;22)Glu255Lysn.t.4
    7M69APYes19.18.4132t(9;22)wtn.t.4
    8F67CPNo27.913.2178t(9;22)wtn.t.10
    9M18APNo3711.8114t(9;22), +8n.t.n.t.13
    10M66BPYes3.47.5121t(9;22), i17, +8Val379Ilen.t.20
    11M74CPNo13.78.7720t(9;22)wtn.t.6
    12F58CPNo28.913.5347t(9;22)n.t.n.t.3
    13M65BPYes96.111.366t(9;22)Thr315Ala Gly250Glu1510
    14M30CPNo156.812.2171t(9;22)wtn.t.12
    15M64CPNo3428.9478t(9;22)n.t.7n.t.
    16F74CPNo103.510.5717t(9;22)n.t.5n.t.
    17M19CPNo9614.6500t(9;22)n.t.618
    18F45CPNo89.112.2536t(9;22)n.t.n.t.12
    19M73APNo112.211.9394t(9;22), t(2;10)n.t.8n.t.
    20M68CPNo190.012.3490t(9;22)n.t.8n.t.
    21M67CPNo41.414.8310t(9;22)n.t.820
    22M73CPYes21.113.7754t(9;22)Phe359Valn.t.12
    23M67APYes16.29.969t(9;22)Met244Val68
    • CP indicates chronic phase; AP, accelerated phase; BP, blast phase; WBC, white blood cell count; Hb, hemoglobin; Plt, platelet count; wt, wild type BCR/ABL; and n.t., not tested.

  • Table 2

    Response of leukemic cell lines to Hsp32/HO-1 inhibitors

    Cell linePEG-ZnPP (μM)SMA-ZnPP (μM)ZnDPPIX (μM)
    IC50RangeIC50RangeIC50Range
    K56232–464–887–10
    KU81252–772–1254–6
    Imatinib-resistant K56243–674–82420–30
    BaF3p210wtn.tn.t165–28n.tn.t
    BaF3p210T315In.tn.t85–10n.tn.t
    BaF3p210E255Kn.tn.t105–13n.tn.t
    BaF3p210Y253Fn.tn.t1510–25n.tn.t
    BaF3p210M351Tn.tn.t135–18n.tn.t
    BaF3p210H396Pn.tn.t65–8n.tn.t
    • Cell lines were cultured in control medium or in various concentrations of Hsp32/HO-1 inhibitors for 48 hours. Thereafter, proliferation of cells was measured by 3H-thymidine incorporation assay. IC50 values (μM) represent the mean from at least 3 independent experiments. The ranges of IC50 values (μM) are also provided.

    • n.t. indicates not tested.

  • Table 3

    Inhibition of clonogenic growth of primary CML cells by SMA-ZnPP

    Control mediumSMA-ZnPP
    CFU-GMBFU-ECFU-GEMMCFU-GMBFU-ECFU-GEMM
    CML (MNC) no. 1291333000
    CML (MNC) no. 2181600000
    CML (MNC) no. 317590000
    Healthy donor (MNC) no. 162711200
    Healthy donor (MNC) no. 293622151
    Healthy donor (MNC) no. 383705351
    Healthy donor (CD34+ cells)431102311061
    CML (CD34+ cells) no. 1121073000
    CML (CD34+ cells) no. 2212100090
    CML (CD34+ cells) no. 318220250
    • Primary mononuclear cells (MNC) were isolated from the peripheral blood of untreated CML patients or from healthy controls by Ficoll density centrifugation and were then cultured in control medium or SMA-ZnPP (10 μM) for 24 hours. Thereafter, cells were cultured in methylcellulose in the presence of cytokines as described in the text for 14 days. Then, cultures were examined for the number of colonies by an inverted microscope. Results show the numbers of colonies per 105 cells (MNC seeded on day 0) or colonies per 5 x 103 CD34+ cells and represent the mean of duplicates in each donor.