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Chem. Res. Toxicol. 2001, 14, 16-24
DNA Topoisomerase II Inhibition by Peroxisomicine A1 and Its Radical Metabolite Induces Apoptotic Cell Death of HL-60 and HL-60/MX2 Human Leukemia Cells Ame´lie Lansiaux,† William Laine,† Brigitte Baldeyrou,† Christine Mahieu,† Nicole Wattez,† Herve´ Vezin,‡ Francisco J. Martinez,§ Alfredo Pin˜eyro,| and Christian Bailly*,† INSERM U-524 et Laboratoire de Pharmacologie Antitumorale du Centre Oscar Lambret, IRCL, Place de Verdun, 59045 Lille, France, Laboratoire de Chimie Organique Physique associe´ a` l’ENSCL, ESA CNRS 8009, USTL Baˆ t. C4, 59655 Villeneuve d’Ascq, France, and Facultad de Medicina, Departamento de Farmacologı´a y Toxicologı´a y Departamento de Medicina Interna, Universidad Auto´ noma de Nuevo Leo´ n, Av Gonzalitos 235, Monterrey 64460, NL, Me´ xico Received July 11, 2000
Peroxisomicine A1 (T-514) is a dimeric anthracenone first isolated from the plant Karwinskia humboldtiana. The compound presents a high and selective toxicity toward liver and skin cell cultures and is currently the subject of preclinical studies as an antitumor drug. To date, the molecular basis for its diverse biological effects remains poorly understood. To elucidate its mechanism of action, we studied its interaction with DNA and its effects on human DNA topoisomerases. Practically no interaction with DNA was detected. Peroxisomicine was found to inhibit topoisomerase II but not topoisomerase I. DNA relaxation and decatenation assays indicated that the drug interferes with the catalytic activity of topoisomerase II but does not stimulate DNA cleavage, in contrast to conventional topoisomerase poisons such as etoposide. Two human leukemia cell lines sensitive or resistant to mitoxantrone were used to assess the cytotoxicity of the toxin and its effect on the cell cycle. In both cases, peroxisomicine treatment was associated with a loss of cells from every phase of the cell cycle and was accompanied by a large increase in the sub-G1 region which is characteristic of apoptotic cells. The cell cycle changes were more pronounced with the sensitive HL-60 cells than with the resistant HL-60/ MX2 cells (with reduced topoisomerase II activity), in agreement with the cytotoxicity measurements. Treatment of HL-60 cells with T-514 stimulated the cleavage of the poly(ADPribose) polymerase by intracellular proteases such as caspase-3. The cytometry and Western blot analyses reveal that peroxisomicine induces apoptosis in leukemia cells. In addition, we characterized a catabolite of peroxisomicine, named T-510R, in the form of a highly stable radical metabolite. The electron spin resonance and mass spectrometry data are consistent with the formation of an anionic semiquinonic radical. The oxidized product T-510R inhibits topoisomerase II with a reduced efficiency compared to the parent toxin and was found to be about 3-4 times less toxic to both the sensitive and resistant leukemia cell lines than T-514. Collectively, the results suggest that topoisomerase II inhibition plays a role in the cytotoxicity of the plant toxin peroxisomicine. Inhibition of topoisomerase II may serve as an inducing signal triggering the apoptotic cell death of leukemia cells exposed to the toxin. The dihydroxyanthracenone unit may represent a useful chemotype for the preparation of topoisomerase II-targeted anticancer agents.
Introduction Peroxisomicine A1, also known as compound T-514 (Figure 1), is a dimeric anthracenone isolated from the fruits and seeds of Karwinskia humboldtiana, a toxic plant (Rhamnaceae) indigenous to desert areas of Southern Texas and northern and central Mexico (1). The toxin has also been found in many other plants of the genus Karwinskia (2), in particular in the fruits of the small * Corresponding author. E-mail:
[email protected]. † INSERM-COL. ‡ CNRS. § Departamento de Farmacologı´a y Medicina Interna, Universidad Auto´noma de Nuevo Leo´n. | Departamento de Farmacologı´a y Toxicologı´a, Universidad Auto´noma de Nuevo Leo´n.
tree Karwinskia parvifolia growing in Mexico (3). Accidental ingestion of the toxic fruits produces flaccid paralysis similar to the Guillain-Barre syndrome and poliomyelitis (4). Experimental acute intoxication with peroxisomicine caused severe damage in the lungs and livers of treated animals as well as important neurological lesions. Because of its high and selective toxicity toward different cell lines, such as primary liver and skin cell cultures, peroxisomicine has been tested as an antitumor drug. Interestingly, neoplastic cells derived from hepatic, pulmonary, and colonic tissues were found to be more sensitive to peroxisomicine than the corresponding normal cells. The selective toxicity of peroxisomicine toward tumor cells was superior to that of conventional antican-
10.1021/tx000145j CCC: $20.00 © 2001 American Chemical Society Published on Web 12/15/2000
Peroxisomicine-Induced Topoisomerase II Inhibition
Figure 1. Structure of peroxisomicine (T-514) [3,3′-dimethyl3,3′,8,8′,9,9′-hexahydroxy-3,3′,4,4′-tetrahydro(7,10-bianthracene)1,1′(2H,2′H)-dione]. An energy-minimized structure of the drug is shown. HyperChem 5.01 and Alchemy 2000 were used to construct the structures. The proposed structure of the oxidized peroxisomicine radical product T-510R is shown.
cer drugs such as mitomycin and vincristine (5). Moreover, this drug exhibited no mutagenic activity in peripheral blood lymphocytes (6). It is now undergoing preclinical screening as an anticancer agent (7, 8). The toxicology data on peroxisomicine are relatively abundant, but in contrast, almost nothing is known concerning its mechanism of action. In analogy with other anthracenic compounds, we thought that the toxin could interact with DNA and perhaps inhibit DNA topoisomerases, as is the case with a number of anticancer drugs. We examined the issue of DNA binding and topoisomerase I and II inhibition by peroxisomicine using complementary biophysical and biochemical methods. The finding that the drug interferes with the activity of human topoisomerase II prompted us to investigate its effect at the cellular level, using human promyelocytic leukemia cells sensitive (HL-60) or resistant (HL60/MX2) to the antitumor drug mitoxantrone which is an inhibitor of topoisomerase II. Peroxisomicine was found to be a potent inducer of apoptosis for HL-60 cells.
Experimental Procedures Chemicals and Biochemicals. Peroxisomicine was isolated and purified as previously described (9). A 10 mM stock solution was prepared in DMSO prior to dilution with water (416 ) 29 250 M-1 cm-1). Because of the limited stability of the drug in DMSO (or ethanol), fresh solutions were prepared for each experiment and used immediately after the drug was dissolved in the solvent. Etoposide and camptothecin were from Sigma Chemical Co. All other chemicals were analytical grade reagents. DNA Relaxation Experiments. Supercoiled pKMp27 DNA (0.5 µg) was incubated with 4 units of human topoisomerase I or II (TopoGen) at 37 °C for 1 h in relaxation buffer [50 mM Tris (pH 7.8), 50 mM KCl, 10 mM MgCl2, 1 mM dithiothreitol, and 1 mM EDTA] in the presence of varying concentrations of peroxisomicine. Reactions were terminated by adding SDS to a final concentration of 0.25% and proteinase K to a final concentration of 250 µg/mL. DNA samples were then added to the electrophoresis dye mixture (3 µL) and electrophoresed in a 1% agarose gel at room temperature for 2 h at 120 V. Gels were stained with ethidium bromide (1 µg/mL), washed, and photographed under UV light. Similar experiments were performed using ethidium-containing agarose gels. The same procedure was used for the kinetoplast assay. Briefly, kDNA (TopoGen) was incubated with the peroxisomicine prior to adding topoisomerase II. After the action of the enzyme, samples were treated with SDS and proteinase K and the DNA species were resolved by electrophoresis on agarose gels.
Chem. Res. Toxicol., Vol. 14, No. 1, 2001 17 Cell Cultures and Survival Assay. Human HL-60 and HL60/MX2 promyelocytic leukemia cells were obtained from the American Tissue Culture Collection. Cells were grown at 37 °C in a humidified atmosphere containing 5% CO2 in RPMI 1640 medium, supplemented with 10% fetal bovine serum, glutamine (2 mM), penicillin (100 units/mL), and streptomycin (100 µg/ mL). The cytotoxicity of the drug was assessed using a cell proliferation assay developped by Promega (CellTiter 96 AQueous one-solution cell proliferation assay). Briefly, 2 × 104 exponentially growing cells were seeded in 96-well microculture plates with various drug concentrations in a volume of 100 µL. After incubation for 72 h at 37 °C, 20 µL of the aqueous soluble tetrazolium dye (10) was added to each well, and the samples were incubated for an additional 2 h at 37 °C. Plates were analyzed on a Labsystems Multiskan MS (type 352) reader at 492 nm. Cell Cycle Analysis. For flow cytometry analysis of the DNA content, 106 HL-60 cells in the exponential growth phase were treated with graded concentrations of peroxisomicine for 24 h and then washed three times with citrate buffer. The cell pellet was incubated with 250 µL of trypsin-containing citrate buffer for 10 min at room temperature and then with 200 µL of citrate buffer containing a trypsin inhibitor and RNase (10 min) prior to adding 200 µL of propidium iodide (PI) at a concentration of 125 µg/mL. Samples were analyzed on a Becton Dickinson FACScan flow cytometer using the LYSYS II software which is also used to determine the percentage of cells in the different phases of the cell cycle. PI was excited at 488 nm and fluorescence analyzed at 620 nm (Fl-3). DEVD-pNA and IETD-pNA Cleavage. DEVD-pNA and IETD-pNA cleavage activities were measured using the ApoAlert CPP32/caspase-3 and ApoAlert caspase-8 assay kits (Clontech, Palo Alto, CA), and the recommended protocols were followed. Briefly, 2 × 106 exponentially growing HL-60 cells in 2 mL of RPMI 1640 medium were treated with peroxisomicine at the indicated concentration for 24 h at 37 °C. Cells were pelleted by centrifugation and resuspended in 50 µL of the lysis buffer. The lysed cell mixture was then incubated on ice for 10 min prior to centrifugation (12 000 rpm for 3 min at 4 °C). Fifty microliters of 2× reaction buffer supplemented with 10 mM dithiothreitol was then added to each tube incubated at 4 °C. During this period, a control was prepared by adding 0.5 µL of 1 mM DEVD-fmk or z-IETD-fmk to a cell sample treated with 0.2 µM staurosporine (24 h at 37 °C). The substrate DEVD-pNA (N-acetyl-Asp-Glu-Val-Asp-pNA) or IETD-pNA (N-acetyl-IleGlu-Thr-Asp-pNA) was added to all tubes (5 µL, 50 µM), and the samples were incubated for 1 h at 37 °C. The formation of p-nitroanilide was assessed at 405 nm using a Labsystems Multiskan MS microtiter plate reader. PARP Cleavage Experiments. Exponentially growing HL60 cells (7 × 105) in a serum-free medium were treated with peroxisomicine at the indicated concentration for 24 h at 37 °C. Cells were pelleted by centrifugation and resuspended in 3 mL of lysis buffer containing 25 mM PBS, 0.1 mM PMSF, and the protease inhibitors chymostatin, leupeptin, aprotinin, and pepstatin A (5 µg/mL each). After centrifugation, the pellet or protein (∼30 µg) was resuspended in the loading buffer containing 50 mM Tris-HCl (pH 6.8), 15% sucrose, 2 mM EDTA, 3% SDS, and 0.01% bromophenol blue. The mixture was sonicated for 30 s at 4 °C and then heated to 100 °C for 3 min. For Western blotting, the cell lysates were fractionated on a 7.5% polyacrylamide gel containing 0.1% SDS and then transferred onto Hybond-C nitrocellulose membranes for 40 min at 150 mA using a semidry transfer system. Membranes were blocked with 10% nonfat milk in PBST [0.1% Tween-20 and 25 mM phosphate buffer (pH 7.4)] for 30 min followed by incubation with antiPARP monoclonal antibody (Clontech) (1:10000 dilution in PBST supplemented with 1% nonfat milk) for 30 min. The blots were washed three times (5 min each with PBST) and incubated with goat anti-mouse IgG conjugated to horseradish peroxidase (Amersham LifeSciences, 1:10000 dilution in PBST containing 1% nonfat milk) for 30 min. After three successive washes with
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Chem. Res. Toxicol., Vol. 14, No. 1, 2001
PBST, the Western blot chemiluminescence reagent from NEN was used for the detection. Bands were visualized by autoradiography. Procaspase-3 Processing. HL-60 cells (0.7 × 106 in 1 mL) were treated with peroxisomicine at the indicated concentration for 24 h at 37 °C. Cells were pelleted by centrifugation at 4 °C and washed twice with phosphate-buffered saline (2 × 3 mL) at 4 °C. After centrifugation, the pellet is resuspended in 25 µL of boiling buffer containing 10 mM Tris-HCl (pH 7.4), 1 mM sodium vanadate, 1% SDS, 0.1 mM PMSF, and the protease inhibitors leupeptin (5 µg/mL), aprotinin (10 µg/mL), and pepstatin A (2.5 µg/mL). The mixture is incubated for 10 min at 4 °C prior to addition of 75 µL of the electrophoresis dye solution (15% sucrose, 50 mM Tris-HCl, 2 mM EDTA, 3% SDS, and 0.01% bromophenol blue). Samples were passed through a 26 gauge needle to reduce the viscosity of the solutions and then boiled to 100 °C for 3 min. For Western blotting, the cell lysates (containing about 30 µg of proteins) were fractionated on a 12.5% polyacrylamide gel containing 0.1% SDS and then transferred onto Hybond-C nitrocellulose membranes (Amersham) for 40 min at 0.8 mA/cm2 using a semidry transfer system. Membranes were blocked with 10% nonfat milk in PBST for 1 h at room temperature (or overnight at 4 °C) followed by incubation with a rabbit polyclonal antibody directed against procaspase-3 (1: 1000, Pharmingen). The antibody was diluted in PBST containing 2% nonfat milk, and membranes were incubated for 4 h in the dark under gentle agitation. Blots were washed three times (15 min each with PBST) and incubated with a sheep anti-rabbit IgG conjugated to horseradish peroxidase (Amersham LifeSciences, 1:10000 dilution in PBST containing 2% nonfat milk) for 1 h. After three successive washes (15 min each) with PBST, the Western blot chemiluminescence reagent from NEN was used for the detection. Product Analysis by Electron Spin Resonance (ESR). X-band ESR spectra were recorded at room temperature using a Varian E-9 spectrometer operating with a magnetic field modulation of 100 kHz, in a TM cavity with a 0.1 mm quartz flat cell. The g values were determined taking the strong pitch (g ) 2.0028) as a standard. A freshly prepared 1 mM solution of peroxisomicine was prepared in a 50:50 (v:v) water/DMSO mixture or in ethanol, and the ESR spectra were recorded at intervals over a period of 16 h. Simulated spectra were obtained with the Bru¨ker Winsimfonia software. Mass Spectrometry. MALDI (mass-assisted laser desorption ionization) mass spectra were recorded on a Finigan MAT Vision 2000 (Bremen). The matrix that was used was dihydroxybenzoic acid/water. For mass spectrometry analysis, peroxisomicine was dissolved in a 50:50 (v:v) water/DMSO mixture and incubated for 48 h. After the reaction, the product was dried under vacuum and dissolved in methanol for mass analysis.
Results DNA Interaction. Peroxisomicine contains a pair of planar anthracenone chromophores (Figure 1), and for this reason, we thought that the drug could intercalate into DNA, as is the case with monomeric anthracenone derivatives (11, 12). This hypothesis turned out to be incorrect. The absorption spectrum of the drug was affected little by the addition of excess DNA. Only a 4% hypochromism was observed in the absorption band centered at 411 nm. Moreover, at a 1:1 drug:base pair ratio, the drug did not modify the thermal denaturation profile of calf thymus DNA or different polynucleotides containing different arrangements of A‚T and G‚C base pairs. In addition, both circular and electric linear dichroism measurements (performed at a DNA:drug ratio of 20 in a low-salt buffer) showed no sign of interaction between calf thymus DNA and peroxisomicine. DNase I footprinting experiments also showed no sequence preference (data not shown). These differents negative results
Lansiaux et al. Table 1. Cytotoxicity (IC50, µM)a peroxisomicine T-514 T-510R etoposide mitoxantrone
HL60
HL60/MX2
RRIb
0.35 1.05 0.07 0.04
1.04 3.55 4.36 0.29
2.97 3.38 62.3 7.25
a The drug concentration that inhibits cell growth by 50% after incubation in liquid medium for 72 h. Each drug concentration was tested in triplicate, and the SE of each point is
