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Mol Cancer Ther. 2006;5:1166-1175
© 2006 American Association for Cancer Research

Research Articles: Therapeutics

Novel triiodophenol derivatives induce caspase-independent mitochondrial cell death in leukemia cells inhibited by Myc

Matilde Parreño1, Jose P. Vaqué4, Isolda Casanova1, Pilar Frade4, M. Virtudes Céspedes1, Miguel Angel Pavón1, Antoni Molins1, Mercedes Camacho2, Luis Vila2, Josep F. Nomdedeu3, Ramon Mangues1 and Javier León4

1 Oncogenesis and Antitumor Drug Group, Laboratori d'Investigació Gastrointestinal 2 Laboratory of Inflammation Mediators, and 3 Department of Hematology, Institut de Recerca Hospital de la Santa Creu i Sant Pau, Barcelona, Spain, and 4 Grupo de Biología Molecular del Cáncer, Departamento de Biología Molecular-Unidad de Biomedicina del CSIC, Facultad de Medicina, Universidad de Cantabria, Santander, Cantabria, Spain

Requests for reprints: Javier León, Departamento de Biología Molecular, Facultad de Medicina, Universidad de Cantabria, Avenida Cardenal Herrera Oria, s/n 39011 Santander, Cantabria, Spain. Phone: 34-942-201952; Fax: 34-942-201945/934-552331. E-mail: leonj{at}unican.es


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
2,4,6-Triiodophenol (Bobel-24, AM-24) was originally described as a nonsteroid antiinflammatory molecule. We have synthesized three derivatives of Bobel-24 (Bobel-4, Bobel-16, and Bobel-30) and tested their activities as putative antileukemic agents. We have found that Bobel-24 and Bobel-16 were dual inhibitors of cyclooxygenase and 5-lipoxygenase, whereas Bobel-4 and Bobel-30 were selective against 5-lipoxygenase. We have tested the antiproliferative activity of these compounds on a panel of cell lines derived from myeloid and lymphoid leukemias (K562, Raji, HL-60, and Molt4). The cytotoxic IC50 in these cell lines ranged between 14 and 50 µmol/L, but it was higher for nontransformed cells such as 32D, NIH3T3, or human leukocytes. All compounds showed cytotoxic activity on all tested cell lines, accompanied by DNA synthesis inhibition and arrest in the G0/G1 phase. Bobel-16, Bobel-4, and Bobel-24 induced a caspase-independent cell death in K562 and Raji cells, accompanied by chromatin condensation, cytochrome c release, and dissipation of mitochondrial membrane potential in a concentration-dependent manner and production of reactive oxygen species. As the proto-oncogene MYC is involved in mitochondrial biogenesis and survival of leukemia cells, we tested its effect on bobel activity. Bobel-24 induced down-regulation of MYC in K562 and, consistently, ectopic expression of MYC results in partial protection towards the cytotoxic effect of Bobel-24. In conclusion, Bobel derivatives induce a caspase- and Bcl-2-independent cell death in which mitochondrial permeabilization and MYC down-regulation are involved. Bobels may serve as prototypes for the development of new agents for the therapy of leukemia. [Mol Cancer Ther 2006;5(5):1166–75]


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Prostaglandins and leukotrienes are signaling mediators generated from arachidonic acid by the action of cyclooxygenases (COX-1 and COX-2) and 5-lipoxygenases (5-LOX), respectively. COX is expressed in most cells, whereas 5-LOX is mainly expressed in inflammatory cells such as polymorphonuclear leukocytes (1). Leukotrienes modulate the growth of several cell types and it has also been shown that various 5-LOX metabolites from arachidonic acid regulate murine and human hematopoiesis in vitro (2). COX and 5-LOX inhibitors are used as antiinflammatory drugs (3). Some 5-LOX inhibitors inhibit the proliferation of malignant human hematopoietic cell lines although they do not seem to affect the growth of normal hematopoietic cells (4). Several reports have described that activation of 5-LOX and increase of its intermediate by-products (hydroperoxides), induce programmed cell death (PCD) in human T cells, neutrophils (5), and Jurkat cells (6). Despite the ability of LOX or COX inhibitors to cause growth inhibition and/or cell death (7–9), the mechanisms by which these inhibitors act and whether their cytotoxic effects could be characterized as apoptosis, necrosis, or other forms of PCD, is unclear. Nevertheless, the fact that the concentrations of 5-LOX or COX inhibitors required to induce cell death are usually higher than those required to inhibit LOX or COX activity, and that these compounds remain active in cells deficient in LOX (9, 10) or COX activity (11), suggest that mechanisms other than LOX or COX inhibition must also be involved in their antiproliferative effects.

MYC oncogene encodes the transcription factor c-Myc (herein termed Myc) that plays a pivotal role in the control of cell proliferation, cell differentiation, cell size, and apoptosis (12–14).

Myc activates genes involved in cell cycle control, energetic metabolism, protein and ribosome synthesis, and other functions. Also, an important number of Myc target genes are repressed by Myc (15).5 In some models, enforced expression of Myc in the absence of growth factors results in apoptosis, whereas in others, Myc acts as a survival factor and is down-regulated in response to treatments causing cell death or growth arrest (16, 17). Recently, it has been shown that Myc is also involved in mitochondrial biogenesis (18).

Consistent with these ex vivo effects, deregulated expression of MYC is found in a wide array of human cancers (13, 19). Leukemia and lymphoma are clear examples of cancers in which MYC deregulation has been described and associated with tumor progression (19–21). Also, transgenic models with Myc deregulated expression in lymphoid tissue or erythroid precursors have shown that MYC induces lymphomagenesis or erythroleukemia, respectively (21, 22). Our previous work also showed a survival effect of Myc on K562, a CML-derived cell line (23).

The present study examines the mechanism by which a new family of synthetic dual 5-LOX and COX inhibitors, named bobels, with different specificities and selectivities toward 5-LOX and COX, induce cell death in leukemia. Our results show that these compounds produce mainly caspase-independent PCD (24). Moreover, cell death is usually accompanied by reactive oxygen species (ROS) production, mitochondrial membrane depolarization, S phase depletion, and Myc down-regulation. These inhibitors are potential drugs for the treatment of leukemia.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture and Reagents
Molt4, HL60, K562, Raji, 32D, and NIH3T3 cell lines were obtained from American Type Culture Collection. NIH3T3 were grown in DMEM and the rest of the cell lines were cultured in RPMI 1640 (Life Technologies, Gaithersburg, MD) supplemented with 10% fetal bovine serum, L-glutamine (2 mmol/L), and penicillin/streptomycin (50 units/mL). The medium for 32D cells was also supplemented with a 10% supernatant of WEHI-3B (as an interleukin-3 source). K562 cell lines carrying Zn2+-inducible Myc (KmycB) or overexpressing Bcl-2 (KBcl2, K562 transduced with Bcl-2 retrovirus) have been described elsewhere (25, 26). Cyclosporin A (CsA) and A23187 were purchased from Sigma (St. Louis, MO). Benzyl-oxycarbonyl-Val-Ala-Asp-fluoromethyl ketone was from Calbiochem (La Jolla, CA). Bobel-24 (2,4,6-triiodophenol, AM-24) was provided by Industrial Farmaceutica Cantabria (Santander, Spain). The synthesis of Bobel-4, Bobel-16, and Bobel-30 is described in Results. Bobels were purified by recrystallization from acetone/water (Bobel-16 and Bobel-30) and by passage through a preparative chromatographic column on SiO2, eluting with a mixture of ethyl acetate and n-hexane (1:1; Bobel-4). The purity of the compounds was tested by TLC. Stock solutions were prepared in DMSO. For the treatments, cells growing at a density of 80,000 cells/mL were treated with the indicated concentrations of bobels for 24 hours. Control cells were treated with the corresponding volumes of the bobel vehicle.

5-LOX and COX IC50 Determination Assays
5-LOX inhibition assay was done in suspensions of human polymorphonuclear leukocytes prepared as described previously (27). Drugs dissolved in DMSO were added to 350 µL of leukocytes suspensions, adjusted to a density of 18 x 106 cell/mL, containing 2 mmol/L CaCl2 and 1.5 mmol/L MgCl2 and kept at 37°C for 5 minutes prior to the addition of substrate and calcium ionophore. The reaction was started by adding 8.33 µmol/L (final concentration) of [14C]-arachidonic acid (56 mCi/mmol, Amersham Biosciences, Buckinghamshire, United Kingdom) and 5 µmol/L of calcium ionophore A23187. After 5 minutes at 37°C, the reaction was stopped by adding 300 µL of cold stop solution (2% acetic acid in methanol), and the samples were kept at –80°C until eicosanoid analysis. Analysis of 5-LOX–derived compounds was done in a reverse-phase high-pressure liquid chromatography column as described previously (27). 5-LOX activity was evaluated as the sum of peak radioactivity counts associated with all eicosanoids formed through the 5-LOX pathway: leukotriene B4, 6-trans-leukotriene B4 epimers, 5,6-dihydroxy-eicosatetraenoic acid epimers, 5-hydroxy-12-methoxy-leukotriene B4, 20-hydroxy-leukotriene B4, and 5-hydroxyeicosatetraenoic acid.

COX inhibition assays were done in human platelet suspensions prepared as described previously (28). Drugs were added to 175 µL of platelet suspensions, adjusted to a density of 170 x 106 platelets/mL and incubated at 37°C for 5 minutes prior to the addition of substrate. The reaction was started by adding 15 µmol/L of [14C]-arachidonic acid (final concentration). After 5 minutes at 37°C, the reaction was stopped as described above and the prostanoids produced were analyzed by reversed phase high-pressure liquid chromatography (27). The COX activity was evaluated as the sum of peak radioactivity counts associated with all eicosanoids formed through the COX pathway: thromboxane B2, prostaglandin F2{alpha}, prostaglandin E2, prostaglandin D2, and 12-hydroxy-eptadecatrienoic acid.

Arachidonic Acid Metabolism of K562 and Raji Leukemic Cell Lines
A 0.5 mL aliquot of a cell suspension at a density of 20 x 106 cells/mL in RPMI (without serum) was incubated at 37°C in the presence of 25 µmol/L [14C]-arachidonic acid for 15 minutes. To test the 5-LOX activity, cells were preincubated with 0.2 mmol/L of acetyl salicylic acid (Sigma) for 30 minutes in RPMI at 37°C to irreversibly inhibit COX activity. Cells (107 cells in 0.5 mL) were washed and incubated with 25 µmol/L [14C]-arachidonic acid and 5 µmol/L calcium ionophore A23187 at 37°C for 15 minutes. The reaction was stopped and the samples were analyzed as described above (27). The drugs at the final concentrations indicated in Results were added to the cell suspensions 20 minutes before the addition of the radioactive substrate, and samples were processed as indicated above.

Cell Viability Assays
Viability assays were done by using the "Cell Proliferation Kit II" (XTT assay, Roche, Germany). Cells were plated at a density of 5,000 cells per well on 96-well plates in 100 µL of culture medium. After 12 hours, the bobel was added to a final concentration between 1 and 150 µmol/L and incubated for 48 hours. After 48 hours of incubation, 50 µL of XTT labeling mixture was added to each well and incubated for an additional 4 hours. The absorbance was determined at 490 nm.

Evaluation of the Cell and Nuclear Morphology by Phase Contrast and Fluorescent Microscopy
Cells were pretreated with 1 ng of Hoechst 33342/mL medium for 24 hours, and later plated in 96-wells plate at 8,000 cells/well in 100 µL of medium for 12 hours. Then, the cells were incubated with different concentrations of compound for 16, 24, or 48 hours. The cell and chromatin morphology was recorded under 20x or 40x magnification in a contrast (bright-field) microscopy and fluorescence microscopy with Axiovert-200M microscope (Zeiss, Jena, Germany).

Cell Cycle Analysis
Cell lines (1.5 x 106 cells in 10 mL of medium) were treated for 24 hours with bobel compounds and then supplied with 1 µmol/L bromodeoxyuridine in complete medium for 1 hour at 37°C. The cells were then fixed in 70% ethanol overnight at 4°C and stained with FITC labeled anti-bromodeoxyuridine antibody (DAKO, Carpinteria, CA) for 30 minutes at room temperature followed by the addition of propidium iodide solution (5 µg/mL). Cells were then analyzed on a Becton Dickinson FACScan instrument using CellQuest software (BD Biosciences, San Jose, CA).

Mitochondrial Membrane Potential Detection
Mitochondrial membrane potential ({Delta}{Psi}) was determined with 5,5',6,6'-tetrachloro-1,1'-tetraethyl-benzimidazolylcarbocyanine iodide reagent (BD MitoScreen Kit, BD Biosciences). Cell lines (0.8 x 106 cells in 10 mL) were harvested after 16 to 20 hours treatment with Bobel compounds or vehicle, centrifuged at 400 x g, and the cell pellet was resuspended in 0.5 mL of 5,5',6,6'-tetrachloro-1,1'-tetraethyl-benzimidazolylcarbocyanine iodide solution and incubated at 37°C for 10 to 15 minutes and analyzed by flow cytometry (Becton Dickinson FACScan instrument). A dot-plot of red fluorescence (FL2) versus green fluorescence (FL1) was drawn. Red fluorescence corresponds to living cells with intact mitochondrial {Delta}{Psi} and green fluorescence to cells with lost {Delta}{Psi}.

ROS Detection
ROS generation was assayed using dihydroethidium probes (Molecular Probes, Invitrogen, San Diego, CA). The reaction is relatively specific for superoxide anion (O2–) production. Cells were harvested after treatment, and incubated with 4 µmol/L dihydroethidium in RPMI medium without fetal bovine serum and without phenol red for 45 minutes. After that, cells were rinsed with medium, the cell pellet was resuspended in 0.5 mL of medium, and immediately analyzed by flow cytometry. The number of counts versus green fluorescence (FL1-Height) is shown.

Preparation of Cytosolic and Mitochondria-Enriched Fractions
To determine mitochondrial cytochrome c release, 0.8 x 106 cells (in 10 mL) treated with bobels were harvested and washed with 150 µL of extraction buffer [20 mmol/L Hepes-KOH (pH 7.5), 10 mmol/L KCl, 1.5 mmol/L MgCl2, 1 mmol/L EGTA, 1 mmol/L DTT, 250 mmol/L sucrose, 100 mmol/L phenylmethylsulfonyl fluoride, 5 µg/mL pepstatin A, 10 µg/mL leupeptin, and 2 µg/mL aprotinin]. The cell pellet was resuspended in 100 µL of buffer and kept on ice for 30 minutes. Then, cells were homogenized using a glass dounce homogenizer and centrifuged twice at 20,000 x g for 30 minutes at 4°C. The pellet obtained represented the nuclear mitochondria–enriched fraction; supernatant represented the cytoplasmic fraction in which cytochrome c is released. Western blot was done from the cytoplasmic fraction.

Western Blot Analysis
Whole protein lysates were obtained as described previously (29). Fifty micrograms of protein cell extracts were resolved by 12% or 15% SDS-PAGE and then transferred to a nitrocellulose membrane. Blots were incubated with the following primary antibodies anti-PARP (Roche Diagnostics, Mannheim, Germany), anti-caspase-3, anti-caspase-7, anti-caspase-8, anti-Myc, anti-Bcl-XL, anti-Bid, anti-cytochrome c (BD PharMingen, San Diego, CA), anti-caspase-9 (Cell Signaling Technology, Beverly, MA), anti-cyclin B1, anti-p21Cip1, anti-actin (Santa Cruz Biotechnology, Santa Cruz, CA), anti-PCNA, anti-cyclin D3, anti-cyclin A, anti-Bax, anti-Bad, and anti-Bcl-2 (BD Transduction, San Diego, CA), anti-glyceraldehyde-3-phosphate dehydrogenase (Chemicon International, Temecula, CA). Secondary antibodies were from Jackson ImmunoResearch Laboratories (Bar Harbor, ME) and the working dilutions were 1:20,000 to 1:10,000. The levels of proteins were visualized by an enhanced chemiluminescence system (SuperSignal; Pierce, Rockford, IL) and autoradiographed.

Northern Blot Analysis
RNAs were prepared with the Trizol reagent according to the manufacturer's instructions (Invitrogen), separated by electrophoresis through an agarose-formaldehyde gel and transferred to nitrocellulose (15 µg of total RNA per lane). The probe for human Myc was a 1.4-kb fragment of human Myc cDNA (30). Probe labeling, with [{alpha}-32P]dCTP, and filter hybridization were carried out according to standard procedures. The amount and integrity of the RNAs were assessed by staining of the filter with ethidium bromide, which reveals 28S and 18S rRNA.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Synthesis and Cytotoxic Activities of Bobel Derivatives
Bobel-4, Bobel-16, and Bobel-30 (Fig. 1 ) were synthesized by iodination at positions 2, 4, or 6 of the corresponding noniodinated phenols carrying the adequate carbon residues or chlorine atoms at positions 2, 3, 4, or 6. Iodination was done by means of an aromatic electrophilic substitution. Bobel-24, Bobel-16, and Bobel-30 are white crystalline solids with quite high fusion points, Bobel-24 (157°C), Bobel-16 (86°C–89°C), and Bobel-30 (137°C–139°C), whereas Bobel-4 is an oil (liquid at room temperature), thus, with a low fusion point (31). Bobel-4 also differs from the other three in that it is easily oxidizable. The isopropyl groups, specific to Bobel-4, have two carbon residues that make this compound more lipophilic.


Figure 1
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  		Figure 1. Chemical structures of bobel compounds. Bobel-24 (AM-24) is the leader of the family. The corresponding chemical names are indicated below the structures. The synthesis of Bobel-4, Bobel-16, and Bobel-30 is described in the text.

 
We analyzed the in vitro inhibitory activity of bobels on 5-LOX and COX. The obtained IC50s are shown in Table 1 . Bobel-4 has the highest selectivity and inhibitory potency on 5-LOX (2.25 µmol/L) although it is less potent as a COX inhibitor. Bobel-16 has the higher inhibitory activity on COX, but also effectively inhibited 5-LOX. Thus, Bobel-16 is a potent dual inhibitor. Bobel-24 (leader compound) was less potent on COX than the other compounds. In summary, Bobel-4 shows 23-fold higher potency toward 5-LOX than to COX, whereas Bobel-30 shows 6.5-fold higher potency and Bobel-24 shows a 2.5-fold higher potency towards 5-LOX than to COX. In this work, we focused on the cytotoxic activities of Bobel-24 (the series leader), Bobel-4, and Bobel-16.


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  		Table 1. Effect of bobels on 5-LOX and COX activities

 
Bobel-16 and Bobel-4 Cause the Lowest Survival on Leukemia Cell Lines
We tested the antiproliferative activity of the bobels on four human leukemia cell lines: K562 (derived from chronic myeloid leukemia), HL60 (derived from acute myeloid leukemia), Raji (derived from B-lymphoblastic leukemia), and Molt4 (derived from T lymphoblastic leukemia). The XTT cell viability assay revealed that bobels were cytotoxic in the micromolar range on all tested cell lines (Table 2 ). However, the antiproliferative IC50 of Bobel-16 was the lowest for the four cell lines (IC50, ~20 µmol/L). This result correlates with the fact that Bobel-16 behaves as a dual COX/5-LOX inhibitor with the highest inhibitor potency on both COX and 5-LOX enzymes. HL60 and Raji cells were the most sensitive towards Bobel-16 treatment (IC50, ~17 µmol/L). The leader compound, Bobel-24, had less antiproliferative activity than the other three derivatives. All these concentrations are clinically relevant because human plasmatic values (Cmax) of 212 µmol/L Bobel-24 (the series leader) can be obtained and are well tolerated (32). We also tested the activity of Bobel-4, Bobel-16, and Bobel-24 on three nontransformed cell types: 32D (murine myeloid cells), NIH3T3 (murine fibroblasts), and human leukocytes from blood donors. In these three cases, the cells were less sensitive to the cytotoxic effect of the Bobel-24 and Bobel-16, whereas the sensitivity to Bobel-4 was similar to K562 and Raji (Table 2). The growth curves for the hematologically derived cells (K562, Raji, 32D, and leukocytes) are shown in Fig. 2A . The curves show the relative resistance of 32D and human leukocytes to the cytotoxic effects of Bobel-16 and Bobel-24.


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  		Table 2. Effect of Bobel-4, Bobel-16, Bobel-30, and Bobel-24 in cell survival of leukemic cell lines (K562, HL60, Raji, and Molt4) and nontransformed cells (32D, human leukocytes, and NIH3T3)

 

Figure 2
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  		Figure 2. Effect of Bobel-4, Bobel-16, and Bobel-24 in cell survival of leukemic cell lines (K562, HL60, Raji, and Molt4). A, cytotoxicity in leukemic cell lines (K562 and Raji), nontransformed myeloid cells (32D), and human leukocytes were determined by the XTT test after 48 h of incubation with Bobel-4, Bobel-16, and Bobel-24 at the indicated concentrations. B, characterization of membrane modification, cell morphology, and chromatin during induction of cell death by 60 µmol/L of Bobel-4 (2-fold, IC50) for 16 h in Raji cells and 40 µmol/L of Bobel-16 (2-fold, IC50) for 24 h in K562 cells. The cell morphology was displayed by contrast microscopy (phase contrast images, top) and nuclear staining by Hoechst staining (fluorescent images, bottom). Original magnification, x20 (Raji) and x40 (K562). Control cells treated with vehicle. C, bobels induce depletion of S phase in leukemic cells. Cell cycle fluorescence-activated cell sorting analysis on cells incubated in the presence of vehicle or 2-fold IC50 of bobel compounds (as stated in Table 2) for 24 h.

 
As Bobel-24 was originally introduced and described as a COX and LOX inhibitor (31, 33), we asked whether these inhibitions were responsible for the cytotoxic activity. We analyzed whether Bobels interfered with the arachidonic acid metabolism by analyzing metabolites of arachidonic acid in K562 (myeloid cell line) and Raji (lymphoid cell line). Cells were labeled in vivo with [14C]-arachidonic acid and its metabolites analyzed by chromatography. We did not detect 5-LOX activity in K562 cells, nor did we find COX activity in Raji cells. In K562 cells, COX activity was inhibited by 72%, 47%, and 50% when cells were treated for 20 minutes with Bobel-24, Bobel-16, and Bobel-4, respectively, at concentrations corresponding to their IC50s (as stated in Table 2). In Raji cells, 5-LOX activity was inhibited by 96%, 2%, and 100% when cells were treated for 20 minutes with Bobel-24, Bobel-16, and Bobel-4, respectively, at concentrations corresponding to their antiproliferative IC50s (as stated in Table 2). In both cell lines, but more dramatic for Raji cells, bobel concentrations that allow half-maximal growth rate (Table 2) resulted in >50% inhibition of COX and 5-LOX activities. Thus, the antiproliferative or cytotoxic effect of the bobels did not correlate with their inhibition of COX or 5-LOX enzymatic activity.

Membrane and Chromatin Modification during Induction of Cell Death by Bobel Compounds
To explore the mechanism by which these compounds decrease cell viability, we first assessed annexin V binding, which detects the degree of phosphatidylserine externalization on the plasmatic membrane, an early marker of apoptosis and late marker of other PCD. The binding of annexin V was small for K562 and Raji after shorter treatment times (<4% after 24 hours; data not shown).

The previous result suggests the induction of a nonclassical apoptotic pathway for the Bobel-induced cell death, either programmed or accidental necrosis. To explore the cytotoxic mechanism of bobels, we investigated whether bobels provoked nuclear condensation and chromatin fragmentation. For this, we stained with the DNA-specific dye, Hoechst. Raji cells were treated with Bobel-4 and K562 cells were treated with Bobel-16 (Fig. 2B). The results showed that both drugs induced chromatin condensation after 16 or 24 hours of treatment. We also observed that bobels induced membrane blebbing at high concentrations (bright-field micrographs in Fig. 2B). We conclude that the mechanism by which these compounds act is not by accidental necrosis because all of them produce chromatin condensation and fragmentation, different levels of annexin exposure and nuclear condensation and membrane blebbing, and all these features are absent in accidental necrosis (34, 35). The cell death is not a consequence of p53 activation by bobels, as K562 and Raji cells carry inactivated p53 alleles (36).

Effect of Bobel Compounds on the Cell Cycle
We next analyzed the apoptosis and cell cycle distribution of K562 and Raji cells in response to bobels through cytometric analysis of propidium iodide–stained cells. These compounds induced an accumulation of cells in the sub-G1 fraction (i.e., with partially degraded chromatin, a cell death indicator) in K562 and, to a lesser extent, in Raji cells, after 24 hours of treatment (Fig. 2C). However, bobels did not provoke chromatin degradation, as assessed by the sub-G1 fraction in 32D cells (Fig. 2C), consistently with its higher resistance to bobel-mediated cell death (Table 2).

On the other hand, flow cytometry revealed that the bobels induced, as expected, a profound repression of DNA synthesis (S phase fraction detected by anti-bromodeoxyuridine-FITC) in K562 and Raji cells (Fig. 2C). We also determined whether some regulatory components of the G1 and M phases of the cell cycle, such as cyclin D3 (the cyclin D with highest expression in K562), cyclin A, and cyclin B, which were down-regulated by bobels in a dose-dependent manner (data not shown).

Bobel-24 and Bobel-16 Induce Caspase-Independent Cell Death in Leukemia Cells
To further investigate the mechanism of cell death induced by the bobels, we analyzed the activation of pro-caspase-3 or caspase-7 by Bobel-24, Bobel-16, or Bobel-4 by Western blot. We did not observe the activation of caspase-3 and caspase-7 at bobel concentrations equivalent to 2-fold IC50 (Fig. 3A ). Also, we did not detect any significant proteolysis of the PARP protein, a known caspase substrate in K562 and Raji (Fig. 3A). Furthermore, we did not detect changes in the levels of the antiapoptotic protein Bcl-2 and Bcl-xL and of the proapoptotic Bax and Bid in both cell lines (data not shown).


Figure 3
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  		Figure 3. A, bobels do not induce executor caspase activation and significant PARP cleavage in K562 and Raji leukemia cell lines. Immunoblot of K562 and Raji cells were done from protein extracts of cells incubated in the presence of vehicle or their corresponding equivalents at 0.5-fold, 1-fold, and 2-fold IC50 for 24 h. Jurkat cells treated with 60 µmol/L etoposide for 6 h were used as a positive control of caspase activation. B, Bobel-24 and Bobel-16 induce the release of cytochrome c. Protein extracts from cells incubated in the presence of vehicle or their corresponding 2-fold IC50s for 24 h. Glyceraldehyde-3-phosphate dehydrogenase was used as a loading control. C, effect of Bobel-16 and Bobel-24 on ROS production. Flow cytometric analysis of ROS content in untreated cells (vehicle) and Bobel-treated cells after 16 h (Bobel-16) or 22 h (Bobel-24) with their corresponding 2-fold IC50s. After treatment, cells were incubated with the probe, 4 µmol/L of dihydroethidium, in free medium for 45 min at 37°C and analyzed with the flow cytometer. As a positive control of ROS production, cells were treated with 15 mmol/L of H2O2 for 1 h. Histograms, events (cell number) versus FL-1 fluorescence intensity (ROS production); thin line, control cells (treated with vehicle alone); dotted line, bobel-treated cells.

 
Thus, these compounds induce a mostly caspase-independent cell death in leukemic cells. We next asked whether the cell death mechanism elicited by bobels could involve the mitochondrial pathway. Thus, we first asked whether the mitochondrial integrity could be affected by the bobel compounds by determining cytochrome c release. The results show that Bobel-24 and Bobel-16 induced cytochrome c release in K562 and Raji cells after 16 hours of treatment (Fig. 3B). This suggests that these bobels affected the mitochondrial permeability.

Effect of Bobels on Mitochondrial Membrane Permeabilization and ROS Production
The finding that bobels alter the mitochondrial membrane permeability of leukemia cells suggests that they could also induce the production of ROS, which is a common mediator of cell death (37). To study whether this was also operative in bobel-mediated leukemic cell death, we analyzed whether ROS production was increased. For this, we stained cells treated with the bobel compounds with dihydroethidium, a cell-permeable fluorescence dye that reacts with a broad spectrum of ROS. ROS production (e.g., oxidized dihydroethidium) was quantified by flow cytometry. As a positive control for ROS production, we determined the relative fluorescent shift in H2O2-treated cells. The viable cells were gated by forward- and side-scatter values. The results showed that treatment of Raji and K562 cells with Bobel-16 and Bobel-24 results in ROS production similar to H2O2-treated cells (Fig. 3C). The results are consistent with a cell death mechanism involving ROS production, at least for Bobel-24 and Bobel-16.

We also analyzed whether bobels induced mitochondrial membrane permeabilization and subsequent membrane depolarization ({Delta}{Psi}), which is associated with most forms of cell death involving the mitochondria. To that purpose, we stained the cells with a fluorescent cationic dye, 5,5',6,6'-tetrachloro-1,1'-tetraethyl-benzimidazolylcarbocyanine iodide, that exhibits potential-dependent accumulation in mitochondria which can be detected by flow cytometry. Bobel-16 in K562 and Raji cell lines induced loss of {Delta}{Psi}, and to a lesser degree, Bobel-24, in K562 cells (Fig. 4A ). In contrast, Bobel-4 hardly induces loss of {Delta}{Psi} in K562 cells, whereas it readily induces loss of {Delta}{Psi} in Raji cells. However, higher levels of Bobel-4, such as 3-fold IC50, in the K562 line, induces mitochondrial depolarization after 16 hours (data not shown).


Figure 4
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  		Figure 4. Effect of Bobel-4, Bobel-16, and Bobel-24 on {Delta}{Psi}. A, the loss of mitochondrial {Delta}{Psi} in Bobel-treated cells is cell type– and compound-dependent. K562 and Raji cells were treated for 16 h with the indicated Bobel compound at concentrations 2-fold IC50 in each case (as stated in Table 2). The cells were then treated for 15 min with 5,5',6,6'-tetrachloro-1,1'-tetraethyl-benzimidazolylcarbocyanine iodide probes and then analyzed by a flow cytometer, as described in Materials and Methods. "+{Delta}{Psi}" (black column) denotes the fraction of cells with polarized mitochondrial membrane and "–{Delta}{Psi}" (gray column) with depolarized mitochondrial membrane. B-24, Bobel-24; B-16, Bobel-16; B-4, Bobel-4; V, control cells treated with vehicle alone. B, CsA reduces bobel-induced depolarization and cell death. Left, pretreatment of cells with 2 µmol/L CsA for 30 min rescues the depolarization of mitochondrial membrane induced by 2-fold IC50 Bobel-16 (B-16) for 16 h. Black and gray columns, similar to (A). Right, pretreatment of cell with 2 µmol/L CsA partially suppresses cell death induced by Bobel-16, as determined by the XTT viability assay. V, control cells treated with vehicle alone. C, z-VAD, a pan-caspase inhibitor, does not increase cell viability in Bobel-24-treated K562 cells. K562 or Jurkat cells were preincubated with or without 25 µmol/L benzyl-oxycarbonyl-Val-Ala-Asp-fluoromethyl ketone for 1 h previous to treatment with Bobel-24 (2x IC50 concentration) or etoposide (60 µmol/L) for 48 h. D, Bcl-2 does not confer resistance to bobel-induced cell death in K562 cells. Immunoblot showing the expression of Bcl-2 of parental K562 and KBcl2 cells treated for 24 h with Bobel-24 (used at a concentration of 2x IC50). The expression of glyceraldehyde-3-phosphate dehydrogenase is also shown to assess protein loading. E, parental K562 and KBcl2 cells were treated with the indicated concentrations of Bobel-4, Bobel-16, and Bobel-24 for 24 h, and cell viability was assayed by the XTT assay. Columns, mean values from four independent experiments; bars, SE.

 
The former results suggest that the bobels could impair the mitochondrial membrane functionality and hence generate ROS. We analyzed whether loss of {Delta}{Psi} is essential for bobel-induced cell death by pretreating the cells with CsA, an inhibitor of the mitochondrial permeability transition pore. For that, we preincubated K562 cells with 2 µmol/L of CsA for 30 minutes followed by their incubation with bobel for 16 hours (Fig. 4B). CsA inhibits the membrane depolarization induced by Bobel-16 in K562 cells (Fig. 4B, left) and partially that induced by Bobel-4 (data not shown) in K562 cells. Moreover, the inhibition of the mitochondria depolarization by CsA correlated with an inhibition of the cell death brought about by Bobel-16, as assayed by the XTT test (Fig. 4B, right). This suggests that the mitochondrial pathway is involved in Bobel-16-mediated cell death, although other mechanisms must also be operative as CsA cannot completely rescue cells from the cytotoxic effect of Bobel-16.

The above data strongly argues for the involvement of mitochondrial-dependent mechanism for the cell death induced by bobels, but were not consistent with a "classical" (caspase-dependent) apoptosis as the main mechanism of cell death. In order to clarify this point, we used two additional approaches: treatment with caspase inhibitors and Bcl-2 overexpression. Figure 4C shows that K562 cells were preincubated with 25 µmol/L benzyl-oxycarbonyl-Val-Ala-Asp-fluoromethyl ketone pan-caspase inhibitor previous to treatment with Bobel-24 and we found that caspase inhibitor did not rescue cytotoxicity at concentrations that were effective on etoposide-dependent Jurkat cell death (a caspase-dependent process; Fig. 3A). Bcl-2 is a mitochondrial outer membrane protein usually associated with resistance to cell death through the mitochondria-caspase pathway. We asked whether Bcl-2 could impair the cytotoxic effect of bobels. We compared the cell viability of K562 and KBcl2, a K562 derivative that express high levels of Bcl-2, which otherwise is not expressed in parental cells (ref. 26; Fig. 4D). The XTT assay showed that overexpression of Bcl-2 did not confer resistance to cytotoxicity induced by Bobel-4, Bobel-16, and Bobel-24 (Fig. 4E), again supporting a caspase-independent mechanism for bobel action.

Bobel Compounds Inhibit Myc Expression as They Induce Cytotoxicity in K562 and Raji Leukemia Cells, and Myc Rescues Bobel-24-Induced K562 Cell Cytotoxicity
The former results indicate a mitochondrial pathway for the cell death mediated by bobels. As described in the Introduction, MYC deregulation is a common finding in leukemia and plays a role in the survival in many leukemia cell lines, including K562 and Raji. Also, Myc stimulates mitochondrial biogenesis. Therefore, we asked whether Myc could modify the cell death mediated by bobels. We first analyzed Myc expression in cells treated with the bobel compounds. K562 and Raji cells were treated for 24 hours with Bobel-24, Bobel-16, and Bobel-4 at different concentrations. We found that, in both cell lines, MYC mRNA expression is down-regulated by Bobel-24 and, to a lesser extent, by Bobel-16, as assessed by Northern blot (Fig. 5A ). The same result was observed at the protein level, as assessed by Western blot (Fig. 5B). MYC down-regulation was more prominent after treating cells longer than 24 hours in all conditions (data not shown). However, Bobel-4, at 2x IC50 concentration did not significantly affect the levels of Myc protein in K562 cells (Fig. 5B), consistent with the divergent chemical and cytotoxic properties of Bobel-4.


Figure 5
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  		Figure 5. Effects of Bobel-24, Bobel-16, and Bobel-4 on MYC expression. K562 and Raji cells were incubated for 24 h with the indicated concentrations of Bobel-24 and Bobel-4, expressed as fold of the corresponding IC50 for each cell line (see Fig. 2A). A, MYC mRNA expression as determined by Northern analysis. The rRNAs on the filter stained with ethidium bromide are shown to assess the loading of RNA. B, Myc protein expression as determined by Western analysis. Expression of {alpha}-actin was used as a loading control. Myc rescues the viability of K562 cells treated with bobel-24. C, MYC expression in KmycB cells. Cells were pretreated for 6 h in the presence of 75 µmol/L of ZnSO4 to induce ectopic Myc expression, then Bobel-24 (used at 75 µmol/L) was added and Myc expression was determined by Northern blot (left) after 36 h and Western blot (right) after 16 h. Left, bigger endogenous and smaller exogenous MYC transcripts in KmycB cells (arrows). D, Myc partially rescues KmycB cells from Bobel-24-induced cell death. KmycB cells were seeded at 1.2 x 105 cells/mL in the presence or absence of 75 µmol/L ZnSO4 for 6 h. Then, cells were plated and incubated in 96-well culture plates with the indicated concentrations of Bobel-24 (left) and Bobel-4 (right) for 16 h and cell viability as determined by the XTT assay. Columns, mean values from four independent experiments; bars, SE.

 
In view of the dramatic down-regulation of Myc elicited by Bobel-24 and also because this is the leader compound, we asked whether Myc overexpression could rescue the cytotoxic effect of Bobel-24. We used the cell line, KmycB, as a tool to address this question. KmycB cells are a K562 subline stably transfected with a MYC expression vector in which the oncogene is under the control of the metallothionein promoter and is inducible by the addition of Zn2+ to the culture medium (25). The results showed that KmycB cells expressed significant levels of exogenous Myc mRNA and protein (Fig. 5C) when treated with Zn2+ or Zn2+ plus Bobel-24. In contrast, Bobel-24 completely abolished endogenous MYC mRNA (Fig. 5C, left) and protein (Fig. 5C, right) expression as expected. We next tested the cytotoxic effect of Bobel-24 on the KmycB cell line in the presence or absence of Myc induction. The results (Fig. 5D, left) showed that the ectopic expression Myc partially rescued KmycB cells from the cytotoxic effect of Bobel-24 (16 hours). As a control, we also tested the effect of Bobel-4 (which hardly decreased the Myc levels in K562, Fig. 5A and B) on the viability of KmycB cells. Consistently, the ectopic expression of Myc did not rescue the viability loss provoked by Bobel-4 in KmycB cells (Fig. 5D, right). Thus, under conditions in which Myc is down-regulated (i.e., with Bobel-24 in K562), Myc ectopic expression antagonized the cytotoxic effect of Bobel-24, whereas in the conditions in which Myc expression is unmodified (i.e., with Bobel-4) ectopic Myc does not modify the cytotoxic response to the compound.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Bobel-24 (2,4,6-triiodophenol, AM-24) was originally designed as a potential antiinflammatory drug with inhibitory activities against COX and LOX. Here, we have found that it exerts a potent cytotoxic activity against several leukemia cell lines. We have designed and described three new derivatives of Bobel-24: Bobel-4, Bobel-16, and Bobel-30. These molecules show different inhibitory selectivities toward 5-LOX and COX, and induce cell death in leukemia cell lines.

Bobel-16 is a dual inhibitor of COX and 5-LOX and shows the highest cytotoxic potency over the studied cell lines. In contrast, Bobel-4 is the most selective against 5-LOX. In many cases, there is no good correlation between the extent of COX or 5-LOX inhibition and the cytotoxic effect on the tested cell lines. Therefore, their cytotoxic mechanism does not seem to be directly related to the inhibition of these enzymatic activities. However, it is important to note that the IC50 of these compounds for all the tested cell lines are within the concentration range clinically attainable (32). Interestingly, Bobel-24 and Bobel-16 show lower cytotoxic activity on nontransformed cells (32D, NIH3T3, leukocytes) than in leukemia cell lines, either from myeloid origin (K562, HL60) or lymphoid origin (Raji, MOLT). This effect is not found for Bobel-4, which presents physicochemical properties divergent from the other bobel compounds.

In the Raji and K562 cell lines, bobels provoke chromatin condensation (pyknosis, nuclear shrinkage), chromatin fragmentation, different degrees of phosphatidylserine exposures, and membrane blebbing. However, we did not detect the activation of pro-caspase-3 or pro-caspase-7. Moreover, a pan-caspase inhibitor or Bcl-2 overexpression does not rescue cytotoxicity by bobels. These features are indicative of PCD (apoptosis-like PCD and necrosis-like PCD) that work mainly through caspase-independent pathways, but it is different from so-called accidental necrosis (34, 38).

Thus, we observed that several cell death mediators are involved in the bobel-induced cell death. They include generation of ROS, dissipation of mitochondrial transmembrane potential ({Delta}{Psi}), and cytochrome c release, which suggest a redox-linked mechanism. Caspase-independent mitochondrial death associated with chromatinolysis has been previously reported (39). Thus, we have shown that Bobel-24 and Bobel-16 induce total or partial depolarization of the mitochondrial membrane in all studied cell lines, and Bobel-4 only in Raji cells or at a concentration >2-fold IC50 on K562 cells, again, consistently with its divergent physicochemical properties. Pretreatment with CsA, an inhibitor of permeability transition pore and hence of the membrane depolarization, was able to partially prevent cell death induced by bobels. Although 5-LOX and COX activity inhibition are not directly responsible for the bobel-induced cell death through the mitochondria, their inhibition could amplify some of the cytotoxic effects produced by these compounds by increasing free arachidonic acid or other lipid second messengers (such as the GL3 ganglioside and ceramide), which are involved in mitochondrial outer membrane permeabilization (38, 40). On the other hand, ROS, the concentrations of which are also increased by cPLA2 and arachidonic acid (38, 41), could also lead to severe mitochondrial dysfunction and produce necrotic PCD or apoptotic PCD. The cytochrome c release and production of arachidonic acid are also involved in the cell death pathways induced by the bobel compounds in pancreatic carcinoma cell lines,6 suggesting that this might be a general mechanism of action of bobels. The reduction in S phase provoked by bobel treatment may be related to the dramatic Myc down-regulation, as Myc is a major regulator of the G1-S transition (14, 15). In agreement with this idea, we observed that the bobel compound provoking a less potent DNA synthesis inhibitory effect on K562 (Bobel-4, Fig. 2C), also provokes a smaller decrease in Myc expression (Fig. 5B).

In many tumor cells, particularly hematologic neoplasias, Myc down-regulation can lead to apoptosis (17, 42). Also, in some models, the cell death mediated by MYC down-regulation is associated with an increase in the production of ROS and cytochrome c release (43–45). Moreover, it has recently been shown that Myc increases mitochondrial mass and function (18).

Our results are in line with these data and suggest that the down-regulation of MYC by bobel compounds induces mitochondrial dysfunction, leading to induction of the downstream events of PCD, such as ROS generation and cytochrome c release. This hypothesis was confirmed by using a K562 cell line with conditional expression of MYC, in which Myc expression remains unabated in the presence of bobel. Under these conditions, these cells show a resistance to the cytotoxic effect of Bobel-24, suggesting that bobels induce cytotoxicity, in part, through the down-regulation of MYC.

In conclusion, this work describes the cytotoxic effect of a new series of molecules on leukemia cells by inducing a caspase-independent cell death. This effect is exerted through the production of ROS, mitochondrial membrane permeability alteration, and MYC down-regulation.


   Acknowledgments
 
We dedicate this paper to the memory of Joaquim Bonal, who pioneered the work on bobels and to whom we remain grateful for his encouragement to do this work. We thank Industrial Farmaceutica Cantabria for Bobel-24. We also thank Mireia Moreno, Josep Úbeda, Luis Carlos Navas, and Mónica Gómez for their technical assistance.


   Footnotes
 
Grant support: Grants SAF02-4193 and 1FD97-1987 from Spanish Ministerio de Educación y Ciencia (J. Leon), SAF03-07437 from Spanish Ministerio de Educación y Ciencia, FIS C03/10 from Ministerio de Sanidad, and AGAUR SGR01050 (R. Mangues) and FIS 01/0853 from Spanish Ministerio Sanidad (M. Parreño). J.P. Vaqué is the recipient of a fellowship from FPI Program (Ministerio de Educación y Ciencia). M. Parreño is partially supported by FIS contract FIS01/3085, Ministerio de Sanidad. R. Mangues is a Researcher of the Catalonian Public Health System.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

5 http://www.myccancergene.org. Back

6 Matilde Parreño, Jose Pedro Vaqué, Isolda Casanova, María Virtudes Cespedes, Miguel Anguel Pavón, Javier León, Ramon Mangues, manuscript in preparation. Back

Received 7/19/05; revised 3/ 7/06; accepted 3/24/06.


   References
Top
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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