Induction of apoptosis by bestatin (ubenimex) in human ... - Nature

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Leukemia (1999) 13, 729–734  1999 Stockton Press All rights reserved 0887-6924/99 $12.00 http://www.stockton-press.co.uk/leu

Induction of apoptosis by bestatin (ubenimex) in human leukemic cell lines K Sekine, H Fujii and F Abe Anticancer Drugs Department, R&D Division, Pharmaceuticals Group, Nippon Kayaku Co Ltd, 31-12, Shimo 3-Chome, Kita-ku, Tokyo, 1150042, Japan

We investigated the growth inhibitory activity of bestatin, an inhibitor of aminopeptidase N (CD13), on six human leukemic cell lines. Proliferation of all the cell lines except KG1 was inhibited by bestatin. P39/TSU, HL60 and U937 were highly sensitive, with 50% growth inhibitory concentrations (IC50) close to the maximum serum concentration when bestatin was orally administered at 30 mg in clinical application. All cell lines except for K562 highly expressed CD13, but a clear correlation between the sensitivity to bestatin and expression of CD13 was not observed. Other aminopeptidase inhibitors such as amastatin A, arphamenine B and WM15 antibody showed no growth inhibitory effects. To confirm the growth inhibitory effects of bestatin, we quantitatively examined DNA fragmentation in five bestatin-sensitive cell lines. Bestatin dose-dependently induced DNA fragmentation in those cell lines. In case of U937, bestatin induced DNA fragmentation quantitatively and DNA ladder and enhanced caspase-3 activity. Furthermore, the growth inhibition by bestatin was reduced by the caspase inhibitor Z-Asp-CH2-DCB. These results suggested that bestatin exhibits direct antileukemic effects against human leukemic cell lines through the induction of apoptosis. Keywords: bestatin; apoptosis; human leukemic cell lines

Materials and methods

Cell cultures Human acute myeloblastic leukemic cell lines P39/TSU and KG1 were obtained from Japanese Cancer Research Resources Bank, Tokyo, Japan. The myeloblastic leukemia cell line HL60 and chronic myeloblastic leukemia cell line K562 were obtained from Riken Cell Bank, Tokyo, Japan. The histiocytic lymphoma cell line U937 was from American Type Culture Collection, Rockville, MD, USA. The acute monocytic leukemia cell line THP1 was from Dainihon Chemical, Osaka, Japan. Cells were maintained in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum from Life Technologies, Rockville, MD, USA and 100 ␮g/ml streptomycin and 100 U/ml penicillin G from Sigma, St Louis, MO, USA in a humidified atmosphere of 5% CO2 and 95% air at 37°C.

Chemicals and antibody Introduction Bestatin ((-)-N-[(2S,3R)-3-amino-2-hydroxy-4-phenyl-butyryl]l-leucine) is a dipeptide obtained from Streptomyces olivoreticuli1 and is known to be a biological response modifier which shows antitumor effects through augmentation of the host immune system.2–7 This molecule inhibits aminopeptidase N, aminopeptidase B and leucine aminopeptidase of mammalian cells.8,9 Aminopeptidase N is identical to the cell-surface antigen CD13,10 which is a myeloid cell lineage marker and is associated with the activation and differentiation of these cells.11,12 Immune-modification of bestatin might be dependent on binding and/or inhibition of aminopeptidases including CD13 on the cell surface.9 Bestatin demonstrated prolongation of disease-free interval and survival period in adult nonlymphocytic leukemia in combination with chemotherapy.13,14 Recently, Ino et al15 reported the immunomodulatory effect of bestatin in lymphoma patients after high dose therapy and autologous bone marrow transplantation (BMT). Recently, higher concentrations of bestatin-induced apoptosis in human non-small-cell lung cancer cell lines have been reported.16 However, there have been few studies of the direct effects of bestatin on leukemic cells. In this study, we demonstrated direct growth inhibition of bestatin in leukemic cell lines in vitro by induction of apoptosis and suggested the possibility that bestatin could clinically have antileukemic effects via apoptosis in addition to immunomodulation.

Bestatin, (-)-N-[(2S,3R)-3-amino-2-hydroxy-4-phenyl-butyryl]l-leucine methyl ester (bestatin methyl ester) and benzyloxycarbonyl-Asp-CH2OC(O)-2,6,-dichlorobenzene (Z-Asp-CH2DCB)17 were prepared by chemical synthesis by Nippon Kayaku Co Ltd, Tokyo, Japan. Amastatin A and arphamenine B were kindly provided by Dr T Aoyagi, Institute of Microbial Chemistry, Tokyo, Japan. FITC-conjugated and non-conjugated WM15 monoclonal antibodies specific for CD13/aminopeptidase N were purchased from Silenus, Hawthorn, Australia. Ac-YVAD-MCA and Ac-DEVD-MCA,18,19 substrates for caspase-1 and caspase-3, respectively, were purchased from Peptide Institute, Osaka, Japan.

Cell growth assay Cells were suspended at a final concentration of 104 cells/ml in RPMI-1640 containing 10% FBS, seeded in microtiter plates (Sumilon, Tokyo, Japan). Then, various concentrations of bestatin were added to each well in triplicate and incubated for specified periods at 37°C in a 5% CO2 atmosphere. After incubation, cells were counted using a Coulter counter (Coulter Corporation, Tokyo, Japan) or by colorimetric WST1 (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)2H-tetrazolium, monosodium salt; Dojindo, Osaka, Japan) assay. WST-1 and 1-methoxy PMS (phenazine methosulfate) mixture were added to the wells, incubated for 2 h then measured with a microplate reader (Dynex, Virginia, USA) using a 450 nm test filter.

Flow cytometric analysis Correspondence: K Sekine; Fax: +81 3 3598 5423 Received 17 August 1998; accepted 18 January 1999

Cells were washed with PBS(−) and suspended in PBS(−) containing 3% FBS and 0.1% NaN3 to 106 cells/100 ␮l, incubated

Induction of apoptosis by bestatin (ubenimex) in human leukemic cell lines K Sekine et al

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with FITC-conjugated WM15 (2.5 ␮g/106) for 30 min on ice and washed twice with PBS(−). FITC-labeled cells were analyzed using a FACScan flow cytometer (Becton Dickinson, Mountain View, CA, USA).

DNA fragmentation assay DNA fragmentation was quantitatively assayed according to the method of Burton et al.20 Briefly, leukemic cells (1– 2 × 106) were lysed in 200 ␮l of 10 mm Tris-HCl (pH 7.4), 10 mm EDTA and 0.5% Triton X-100 (lysate buffer) and incubated at 4°C for 10 min. The lysates were centrifuged at 19 500 g for 20 min. The DNA fraction in the supernatant was defined as free (soluble) DNA, while that in the precipitation was defined as internucleosomal (insoluble) DNA. The precipitates including internucleosomal DNA were lysed in 200 ␮l of lysate buffer again. To both DNA fractions 200 ␮l of 1 N HClO4 was added, incubated at 4°C for 30 min and centrifuged at 19 500 g for 20 min. The precipitates were suspended in 50 ␮l of 1 N HClO4, heated at 70°C for 20 min. After cooling, 100 ␮l of diphenylamine reagent (150 mg diphenylamine, 150 ␮l H2SO4, 50 ␮l 0.2% CH3CHO, 10 ml CH3COOH) was added, incubated at 25°C overnight in dark. The colors were measured with a microplate reader using a 600 nm test filter. DNA fragmentation rate was calculated from the salmon sperm DNA standard and as following. DNA fragmentation rate (%) = soluble DNA × 100. total DNA (soluble DNA + insoluble DNA) DNA ladder was analyzed according to the method described by Mashima et al.17 Briefly, PBS(−)-washed 106 cells were suspended in 20 ␮l of 50 mm Tris-HCl (pH 8.0), 10 mm EDTA, 0.5 mg/ml proteinase-K and incubated at 50°C for 1.5 h. Then, 10 ␮l of 1 ␮g/ml RNase A solution in 10 mm Tris-HCl (pH 8.0), 15 mm NaCl was added. After a further 1.5-h incubation, 10 ␮l of sample buffer containing 10 mm EDTA, 1% low melting point agarose, 0.25% bromophenol blue and 40% sucrose was added, followed by electrophoresis in 2% agarose gels.

Kinetic for caspase activity Caspase activity was measured according to the method of Nicholson et al.18 Cytosolic extracts were prepared from U937 cells by homogenizing PBS(−)-washed cell pellets in 10 mm HEPES-KOH (pH 7.4), 2 mm EDTA, 0.1% CHAPS, 5 mm dithiothreitol, 1 mm PMSF to 107 cells/ml and recovering the post19 500 g supernatant after centrifugation. The cytosolic proteins (5 ␮g/sample) were incubated at 37°C in 20 ␮m amino acid-MCA (4-methylcoumarine-7-amide), 20 mm HEPES-KOH (pH 7.4), 2 mm dithiothreitol and 10% glycerol, and the amount of AMC (7-amino-4-methylcoumarine) was measured with a Baxter Fluorescence Concentration Analyzer (excitation, 365 nm; emission, 450 nm; Baxter, Mandolin, IL, USA). The cleavage activity was calculated from the fluorescence of AMC standard.

Statistical analysis Significant differences were determined by Student’s t-test.

Results

Expression of CD13 on human leukemic cell lines and the sensitivity of cell growth to bestatin We examined whether the inhibitory activity of bestatin to leukemic cell growth in vitro was correlated with the expression of CD13 on the tested leukemic cell lines (Table 1). When the inhibitory activity was examined for 96-h culture, P39/TSU, HL60, U937, THP1 and K562 showed inhibition of proliferation by bestatin. P39/TSU, HL60 and U937 were especially sensitive with IC50 values of 0.48, 0.67, 2.47 ␮g/ml, respectively. These concentrations were close to the maximum serum concentration (2.2 ␮g/ml) when bestatin was clinically administered at 30 mg.21 To investigate the kinetics of the growth inhibitory effects of bestatin, U937 cells were incubated with bestatin for 24, 48 or 96 h. The growth inhibitory effects depended on the treatment time and the values of IC50 were 10.3, 3.3 and 1.2 ␮g/ml, respectively (data not shown). All of the cell lines except K562 highly expressed CD13, on the other hand K562 weakly expressed it (Table 1). KG1 that highly expressed CD13 showed little growth inhibition by bestatin. Thus, the cell lines tested were not suitable to clearly determine the correlation between the growth inhibitory effects of bestatin and the intensity of CD13 expression.

Inhibition of U937 cell growth by bestatin and other aminopeptidase inhibitors We investigated the growth inhibitory effects of other aminopeptidase inhibitors such as amastatin A,22 arphamenine B23 and WM1524 monoclonal antibody, which have been reported to inhibit various aminopeptidase activities (Table 2). However, these inhibitors showed no growth inhibitory effects.

Apoptosis induction leukemic cell lines by bestatin In the above experiments of the cell growth inhibition by bestatin, we observed the morphological changes and found Table 1 Cell-surface expression of CD13 and growth inhibition by bestatin in human leukemic cell lines

Cell line P39/TSU HL60 U937 THP1 K562 KG1 a

Type

Acute myeloblastic leukemia Myeloblastic leukemia Histiocytic lymphoma Acute monocytic leukemia Chronic myeloblastic leukemia Acute myeloblastic leukemia

CD13a

IC50 (␮g/ml)b

40.0 15.1 64.3 20.0 1.2 19.4

0.48 0.67 2.47 6.41 11.61 ⬎100

Expression of CD13 on the cells was visualized by staining with FITC-labeled anti-CD13 antibody and analyzed using a FACScan flow cytometer. The values shown are ratios of the median fluorescence intensity between stained and non-stained cells. b IC50 values are the concentrations of bestatin that induced 50% growth inhibition compared to untreated controls. The leukemic cells were suspended at 104 cells/ml and treated with various concentrations of bestatin in triplicate wells. After 96-h incubation, cells were counted with a Coulter counter and IC50 values were determined.

Induction of apoptosis by bestatin (ubenimex) in human leukemic cell lines K Sekine et al

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Table 2 Effects of various aminopeptidase inhibitors on proliferation of U937 cells

Inhibitor

Amastatin A

Arphamenine B Bestatin

WM15 antibody

Specificity

IC50 (␮g/ml)a

Aminopeptidase N Aminopeptidase A Leucin aminopeptidase Aminopeptidase B

⬎100

Aminopeptidase N Aminopeptidase B Leucin aminopeptidase Aminopeptidase N

2.47

⬎100

⬎25

a

The inhibitory effects on U937 cell growth were examined as indicated in Table 1.

that the cells showed cytoplasmic shrinkage and membrane blebbing suggesting apoptosis (data not shown). To confirm the growth inhibitory effects by apoptosis following bestatin treatment, we quantitatively examined DNA fragmentation in five bestatin-sensitive cell lines (Figure 1). In the tested cell lines, DNA fragmentation was detected by treatment for 96 h with bestatin at various levels. U937, HL60 and THP-1 were intensely induced DNA fragmentation, whereas P39/TSU was induced a little. Furthermore, DNA fragmentation of U937 was quantitatively analyzed every 12 h (Figure 2). The soluble (fragmented) DNA increased in time- and dose-dependent manner. Similarly, distinct DNA ladder was detected by treatment for 48 h with bestatin and the extension of culture time to 96 h strongly induced DNA fragmentation at a lower dose (Figure 3). We examined the involvement of caspase activity in the cytosol of bestatin-treated U937 cells (Figure 4). Caspase-3like activity was enhanced by bestatin in a dose- and timedependent manner, and reached a peak at 24 h after bestatin treatment. Bestatin methyl ester, a more hydrophobic deriva-

Figure 1 Quantitative DNA fragmentation of human leukemic cell lines following bestatin treatment. Cells were suspended to a final concentration of 104 cells/ml and added vehicle (쏔), 10 (`) and 100 (쐽) ␮g/ml of bestatin for 96 h. DNA were extracted as described in Materials and methods and incubated with diphenylamine reagent at 25°C overnight in the dark, then the amounts of DNA were measured with a microplate reader using a 600 nm test filter.

Figure 2 Quantitative DNA fragmentation of U937 cells following bestatin treatment. U937 cells (104 cells/ml) were added vehicle (쎲), 10 (왖), 100 (쑗) ␮g/ml of bestatin and quantitatively analyzed every 12 h as indicated in Figure 1.

Figure 3 DNA fragmentation of U937 cells following bestatin treatment. U937 cells were suspended to a final concentration of 104 cells/ml. Then, bestatin was added at various concentrations and incubated for 48 and 96 h. After incubation, the DNA of U937 cells was prepared as described in Materials and methods and analyzed in 2% agarose gels.

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Figure 4 Activation of caspase-1 and caspase-3 in U937 cells by bestatin. U937 cells (104 cells/ml) were treated with vehicle (쎲), 10 (왖), 100 (쑗) ␮g/ml of bestatin or 1 ␮g/ml bestatin methyl ester (왕). Cytosolic extracts were prepared from U937 cells incubated for various periods. Cytosolic protein (5 ␮g/sample) was incubated with Ac-DEVD-MCA (a) or Ac-YVAD-MCA (b), and the amounts of AMC were measured. The cleavage activity was calculated from the fluorescence of AMC standard and is shown as pmol/min/5 ␮g protein.

tive of bestatin, induced activation of caspase-3 more rapidly and at lower concentrations than bestatin. However, no changes were observed in caspase-1-like activity following treatment with bestatin or its methyl ester. Z-Asp-CH2-DCB is a selective inhibitor of the caspase family.17 Treatment with Z-Asp-CH2-DCB reduced the growth inhibition by bestatin (Figure 5). Discussion In this study, bestatin exhibited growth inhibitory effects on human leukemic cell lines in a dose- and time-dependent

Figure 5 Reduction of bestatin-induced growth inhibitory effects by Z-Asp-CH2-DCB. U937 cells (104 cells/ml) were incubated for 1 h in the absence (쏔) or presence (쐽) of 10 ␮g/ml Z-Asp-CH2-DCB. Bestatin was then added at various concentrations and further incubated for 24 h. After incubation, cell number was determined by colorimetric WST-1 assay. Error bars indicate s.d. *P ⬍ 0.01, in the absence vs presence of 10 ␮g/ml Z-Asp-CH2-DCB.

manner. Bestatin is thought to exhibit its antileukemic effects via immunomodulation related to inhibition of aminopeptidases expressed on the cell surface.8,9 Recently, Kanayama et al25 reported that IL-8 was degraded by aminopeptidase N and Shibuya et al (unpublished data) found that other cytokines including IL-2, IFN-␥ and G-CSF were degraded by aminopeptidases and bestatin showed extension of the half-lives of the cytokines by inhibiting aminopeptidase N. Immunomodulation by bestatin involves the inhibition of degradation or processing of cytokines by aminopeptidases in addition to the various effects of bestatin on immune cells as reported previously.2–7 On the other hand, Murata et al26 reported that U937 cells were differentiated to macrophages by bestatin following inhibition of proliferation. The mechanism of the inhibitory effects on leukemic cells has not yet been elucidated. Therefore, the correlation between the expression of CD13 (aminopeptidase N) on leukemic cells and sensitivity to bestatin was investigated. All cell lines examined except K562 highly expressed CD13 and the sensitivity of growth inhibition by bestatin differed between cell lines. Although KG1 cells expressed CD13 they were almost insensitive to bestatin. Furthermore, WM15 that is an anti-aminopeptidase N monoclonal antibody with inhibitory activity and other aminopeptidase inhibitors did not suppress U937 cell growth. Thus, CD13 expression may be partially involved in the sensitivity to bestatin, but no clear correlation between CD13 expression and bestatin sensitivity was observed. P39/TSU, HL60 and U937 exhibited high sensitivity to bestatin, with IC50 values close to the maximum serum concentration (2.2 ␮g/ml) when bestatin was clinically administered as a single oral dose of 30 mg. These results suggested that the antileukemic effects of bestatin in humans might involve direct growth inhibitory effects against leukemic cells in addition to immunomodulation. Human leukemic cell lines showed morphological changes including cytoplasmic shrinkage and membrane blebbing by bestatin treatment. In addition to these morphological changes, DNA ladder formation in the nucleosomes was dosedependently observed in five bestatin-sensitive cell lines (P39/TSU, HL60, U937, THP1, K562), suggesting that the changes occurred via apoptosis. In these cell lines, HL60,

Induction of apoptosis by bestatin (ubenimex) in human leukemic cell lines K Sekine et al

U937 and THP1 were intensely induced DNA fragmentation, but P39/TSU and K562 were a little. This might indicate that a mechanism of the growth inhibitory effects on P39/TSU and K562 cells by bestatin is not only apoptosis. Furthermore, members of the caspase family including caspase-1 and caspase-3 are activated sequentially during the early phase of apoptosis in the cytoplasm.27 We investigated the activation of caspases by bestatin in U937 cells and found that caspase3 activity gradually increased for 24 h, and decreased thereafter. On the other hand, caspase-1 activity did not change with bestatin treatment. Kuida et al28 reported that caspase-1deficient mice developed apoptosis normally, and Mashima et al29 reported that caspase-1 activity was not involved on etoposide-induced apoptosis in U937 cells. These observations and the results presented here suggested the activation of caspase-1 in apoptosis could be dependent on cell type or on apoptotic stimuli. Moreover, induction of apoptosis by bestatin was inhibited in the presence of Z-Asp-CH2-DCB, a selective inhibitor of the caspase family, suggesting that the caspase family may be involved in the mechanism of bestatin-induced apoptosis. The inhibition of apoptosis by Z-Asp-CH2-DCB is a common phenomenon induced by other antitumor agents.30 Bestatin methyl ester, a derivative of bestatin and aminopeptidase inhibitor, rapidly induced activation of caspase-3 and apoptosis at lower concentrations than bestatin. As bestatin methyl ester is more hydrophobic than bestatin and can readily permeate into the cells, these results suggested that bestatin induces apoptosis by entering the cells and modifying some molecules and involved intracellular aminopeptidase(s). Recently, Constam et al31 reported that puromycin-sensitive aminopeptidase (PSA) is involved in induction of apoptosis by bestatin, indicating that bestatin may induce apoptosis via PSA or other as yet unidentified unknown molecules other than CD13. In conclusion, the inhibitory effects of bestatin on the proliferation of human leukemic cell lines were caused by the induction of apoptosis in a dose- and time-dependent manner. Therefore, for clinical application, bestatin should be administered at higher doses or more frequently than the present dose of 30 mg/day.

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