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Cell and Tumor Biology |
Departments of 1 Chemical Sciences and Technologies and 2 Biology, University of Rome "Tor Vergata"; 3 Department of Drug Research and Evaluation, Section of Pharmacogenetics, Drug Resistance and Experimental Therapeutics, Istituto Superiore di Sanità; and 4 Children's Hospital IRCCS "Bambin Gesù," Rome, Italy
Requests for reprints: Anna Maria Caccuri, Department of Chemical Sciences and Technologies, University of Rome "Tor Vergata", Viale della Ricerca Scientifica, 00133-Rome, Italy. Phone: 39-06-72594378; Fax: 39-06-72594328; E-mail: caccuri{at}uniroma2.it.
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-glutamyl-S-[benzyl]cysteinyl phenyl glycyl diethyl ester; refs. 11–18). The former (ethacrynic acid) binds to the H-site but has low affinity for the enzyme, and conjugated with GSH, it is easily excreted from the cell. Moreover, phase I clinical trials have shown severe toxicity of ethacrynic acid, including diuresis, metabolic abnormalities, and myelosuppression (7). The latter (TER 199) is a glutathione peptidomimetic which rapidly enters cells, and being a prodrug, is activated by the intracellular esterases. A different class of cytotoxic GSH analogues consists of molecules that do not inhibit GST but take advantage of its catalytic power to be activated (7, 19). An example is TER 286, whose structure has the GST binding capacity of a GSH-peptidomimetic molecule. When activated by isoenzymes of the Pi and Alpha classes, the latent cytotoxin TER 286 releases an analogue of cyclophosphamide with inherent cytotoxic activity (20). To avoid the inconvenience of the inhibitor extrusion from the cell by specific pumps, we have recently synthesized and characterized non–GSH-peptidomimetic derivatives of 7-nitro-2,1,3-benzoxadiazole (NBD), which resulted in a strong inhibition of GSTs.5
In the present work, we show the cytotoxic activity of four NBD derivatives on cell lines deriving from different sources, describing in detail the apoptotic process induced by the selected derivative 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX) in human leukemic K562 and CCRF-CEM cell lines. The new GST inhibitor triggers apoptosis by the activation of the JNK/c-Jun–mediated pathway.
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Materials and Methods |
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NBD derivatives were synthesized as reported by Ricci et al.5 Stock solutions of these compounds (50 mmol/L) were prepared in DMSO. Just before use, stock solutions were diluted to the appropriate concentration in RPMI cell medium with final DMSO concentration not exceeding 0.05% to 0.1%. In all experiments, control samples were exposed to requisite DMSO concentration; 0.05% to 0.1% DMSO had no cytotoxic effect.
An evaluation of cell viability at different drug concentrations was determined by sulforodamine B (SRB, Sigma) assay (21). The cells were placed in 96-well microtiter plates at a density of 1.5 x 104 cells per well, in 100 µL of medium. After 24 hours, the cells were exposed to each of the NBD derivatives under study, at the required concentration and allowed to incubate for 48 hours. After incubation, the cell growth was evaluated by an in situ cell fixation procedure followed by a coloring procedure with SRB capable of specifically binding to proteins.
The dose-response profile obtained fulfils the LC50 value (the concentration used to obtain 50% cellular mortality) for each NBD derivative assayed.
In all experiments reported later on, NBDHEX was the selected compound used to investigate the process of cell death in the leukemic CCRF-CEM and K562 cell lines.
NBDHEX was used at a concentration five times the LC50 value of each cell line which corresponds to 2 and 10 µmol/L for CCRF-CEM and K562, respectively. For the apoptosis studies, cells were seeded in culture flasks at 0.8 x 106 cells/mL and after 24 hours, treated with the suitable amount of NBDHEX.
Test of acute toxicity in vivo. The test used 25 males of the BDF1 mouse strain (18-20 g, Charles River Lab., Lecco, Italy) divided into five groups of five mice each. Three groups were treated with NBDHEX in a single administration, through i.p. injection, at the following concentrations: (a) 125, (b) 25, (c) 5 mg/kg. The remaining two groups were used as controls and were given the following: (a) olive oil vehicle containing 2.5% DMSO and (b) a physiologic solution. Mice were monitored for 15 days, providing them with food (RF-18, Mucedola, Italy) and water ad libitum, recording the weight variations of each mouse. Temperature (20 ± 2°C) and humidity (55% ± 5%) were continuously monitored. Treatments and experimental handling of animals were done according to the EU Directive (86/609) and Italian law (D.Lvo 116/1992). A veterinary surgeon was present to check the health status of the animals to avoid physical injury, suffering, and distress. All members of the staff involved in the experiments were trained in working with mice. At the end of this period, animals were bled under light anesthesia through the retro-orbital vein. Blood was collected in vacutainer (Becton Dickinson, Belgium) containing heparin and analyzed immediately after bleeding. The cell count was done by using hemocytometer Sysmex XT2000i (Dasit, Italy) and confirmed by microscopic analysis after May-Grumwald Giemsa staining. Mice were then humanely sacrificed and liver and spleen of each mouse were checked in terms of weight variation. Organs were 10% buffered formalin fixed for 24 hours. Samples were alcohol dehydrated and paraffin embedded. Finally, 4-µm-thick sections were H&E stained.
Enzyme activities. GST activity was measured in total cell lysates prepared as follows: cancer cell lines were collected by centrifugation at 700 x g for 5 minutes at 4°C and washed twice in PBS. Cells were resuspended in PBS and lysated by a 10-second sonication. Lysates were centrifuged at 13,000 x g for 20 minutes at 4°C and aliquots of the supernatant were used to measure the GST activity. Activity was tested spectrophotometrically at 340 nm [where the product of reaction, S-(2,4-dinitrobenzene)-glutathione, absorbs 
= 9.6 (mmol/L)–1 cm–1] and at 25°C, in 0.1 mol/L potassium phosphate buffer (pH 6.5), containing 1 mmol/L EDTA, 1 mmol/L GSH, and 1 mmol/l 1-chloro-2,4-dinitrobenzene (Sigma; ref. 22). One unit of GST activity is defined as the amount of enzyme catalyzing the formation of 1 µmol/min of product at 25°C.
Caspase activity was measured on K562 and CCRF-CEM cell lines treated as follows: pellets of cells obtained at different times of incubation with NBDHEX as described above, were resuspended with lysis buffer [100 mmol/L HEPES (pH 7.5), 0.1% CHAPS, 1 mmol/L EDTA, 1 mmol/L phenyl-methyl-sulfonyl-fluoride, and 10 mmol/L DTT] for 20 minutes on ice. Cells were then disrupted by a 10-second sonication. Lysates were then centrifuged at 13,000 x g for 20 minutes at 4°C and aliquots of the supernatant assayed for caspase activity with the model fluorescent peptide N-Acetyl-Asp-Glu-Val-Asp-7-amido-4-trifluoromethylcoumarin (Ac-DEVD-AFC, Sigma). Proteolytic cleavage of the peptide resulted in a fluorescence emission at 505 nm (excitation at 400 nm). As a negative control, lysates were incubated 30 minutes at 30°C with the caspase inhibitor N-Acetyl-Asp-Glu-Val-Asp-al (Ac-DEVD-CHO, Sigma) before evaluation of enzyme activity.
-Glutamyltranspeptidase (GGT, commercial purified enzyme from Sigma) activity was measured essentially as reported by A. Meister et al. (23) by using 1 mmol/L L--glutamic acid -(3-carboxy-4-nitroanilide) (Sigma) and 20 mmol/L glycylglycine (Sigma) as substrates The assay was done both in 0.1 mol/L Tris-HCl buffer (pH 8.0) and in 0.1 mol/L potassium phosphate buffer (pH 6.5). The effect of NBDHEX on GGT activity was evaluated by incubating the assay solution with different concentrations of NBDHEX from 2 to 20 µmol/L, both in the absence and in the presence of 5 mmol/L GSH. -Glutamyl-cysteinyl-synthetase (GCS) activity was determined on cytosol from K562 cells treated with 10 µmol/L NBDHEX. At different incubation times from 15 minutes to 6 hours, cells were lysed and the supernatant was used for GCS determination essentially as previously reported (24). The effect of NBDHEX on GCS activity was also evaluated at pH 6.5 and 8.0 by adding to the assay solution different concentrations of NBDHEX from 2 to 20 µmol/L, both in the absence and in the presence of 5 mmol/L GSH.
Protein concentration was determined by the bicinchoninic acid protein assay reagent (Pierce, Rockford, IL).
Fluorescence microscopy and flow cytometry. Cells were treated for 24 hours with NBDHEX and apoptosis was detected with the fluorescence microscope by analyzing the nuclear fragmentation after staining with the DNA-specific dye Hoechst 33342 (Sigma).
The percentage of viable cells, early apoptotic and necrotic cells was determined by simultaneous staining of cells with propidium iodide (PI) and with Annexin V (Annexin V-FITC) dye (Sigma). After 12 and 24 hours of treatment, cells were washed twice with cold PBS and resuspended in binding buffer (HEPES supplemented with 25 mmol/L CaCl). Cells were then incubated with Annexin V-FITC (0.5 µg/mL) and PI (2 µg/mL) for 15 minutes at room temperature in the dark. Stained cells were analyzed by a FACScalibur instrument (Becton Dickinson, San Josè, CA) as previously reported (25). The emission of unstained cells treated with NBDHEX was used as background fluorescence.
Flow cytometric data were statistically analyzed by WinMDI version 2.8 software.
Western blot analyzes. K562 and CCRF-CEM cells were treated with 10 and 2 µmol/L NBDHEX, respectively. At each time point analyzed, the cell pellet was washed in PBS and resuspended in lysis buffer containing 10 mmol/L Tris-HCl (pH 7.4), 5 mmol/L EDTA, 150 mmol/L NaCl, 0.5% IGEPAL CA-630, and protease inhibitors (Sigma). After a 30-minute incubation on ice, cells were disrupted by a 10-second sonication. Lysates were then centrifuged at 13,000 x g for 20 minutes at 4°C and supernatants were remo ved and stored at –80°C. Proteins (20 µg) were loaded on 10% SDS-polyacrylamide gel and transferred onto a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA). Polyclonal anti-c-Jun and anti-JNK (1:1,000; Upstate Biotechnology, Lake Placid, NY), anti-phospho-activated c-Jun and JNK isoforms (1:500; Santa Cruz Biotechnology, Santa Cruz, CA), antiactin (1:5,000; Sigma) were used as primary antibodies. Alternatively, the cell pellet was resuspended in lysis buffer containing 62.5 mmol/L Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 50 mmol/L DTT, 0.01% bromophenol blue, and sonicated for 15 seconds to shear DNA and reduce sample viscosity. Sample (20 µg) was loaded on 10% SDS-polyacrylamide gel and transferred onto a nitrocellulose membrane. Polyclonal anti-total (Santa Cruz Biotechnology), phospho-isoform of p38MAPK (Cell Signaling Technology, New England Biolabs, Beverly, MA; 1:1,000), and antiactin (Sigma; 1:5,000) were used as primary antibodies. The specific protein complex formed upon appropriate secondary antibody (Bio-Rad) treatment (1:10,000) was identified using the "SuperSignal" substrate chemiluminescence reagent (Pierce).
Immunoprecipitation. Immunoprecipitations were done as previously described (26). Briefly, 300 µg of protein from total cell lysates were incubated in lysis buffer with 10 µL of anti-JNK antibody to a total volume of 300 µL for 2 hours at 4°C. Immunocomplexes were adsorbed with 20 µL of protein A-Sepharose for 30 minutes at 4°C. Immune pellets were boiled in SDS sample buffer. Proteins (50 µg) were loaded on 15% SDS-polyacrylamide gel and transferred to nitrocellulose. Polyclonal anti-GSTP1-1 (1:1,000; Calbiochem-Novabiochem, Darmstadt, Germany), and anti-JNK antibodies were used as primary antibodies and recognized by the "SuperSignal" substrate chemiluminescence reagent.
Tripeptide glutathione determination. To evaluate the intracellular levels of both reduced and oxidized GSH, cytosol of K562 and CCRF-CEM cells was analyzed at different times (0-8 hours) from their NBDHEX treatment. Intracellular GSH was assayed upon formation of S-carboxymethyl derivatives of free thiols with iodoacetic acid followed by the conversion of free amino groups to 2,4-dinitrophenyl derivatives by the reaction with 1-fluoro-2,4-dinitrobenzene (Sigma) as described by Reed et al. (27). Briefly, the cell suspension was washed with PBS, resuspended and lysated by repeated cycles of freezing and thawing under liquid nitrogen. Lysates were used for GSH/oxidized glutathione (GSSG) assay by the high-performance liquid chromatograhy technique with a NH2 BondPack column (Waters, Milford, MA), after derivatization with 1-fluoro-2,4-dinitrobenzene. Data are expressed as nmoles of GSH equivalents/mg of protein.
Detection of reactive oxygen species. Detection of intracellular ROS was done at different times from NBDHEX treatment, by a 20-minute preincubation of cells with 10 µmol/L 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA, Molecular Probes, Eugene, OR). After ROS-mediated oxidation, the 2',7'-dichlorofluorescein becomes fluorescent and can be detected by flow cytometry after subtraction of the background fluorescence of unstained cells treated with NBDHEX.
Inhibition of the 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol–induced apoptosis. Cells were preincubated with the antioxidant 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxilic acid (TROLOX; OXIS International, Portland, OR) 1 hour before the NBDHEX treatment. TROLOX was used at a concentration of 2 mmol/L, which under our experimental conditions, does not result in toxicity. After 1 hour from the TROLOX addition, cells were treated with NBDHEX and the time course of apoptosis was followed up to 24 hours. The effect of TROLOX on the intracellular ROS generation was determined after 20 minutes from the NBDHEX treatment (when the maximum ROS amount is detected), as reported above.
Apoptosis was also analyzed in K562 cells pretreated with 10 µg/mL of cycloeximide for 1 hour before the addition of NBDHEX to inhibit protein synthesis. Owing to the toxic effect of cycloeximide, we were unable to do experiments with CCRF-CEM.
Finally CCRF-CEM and K562 cell lines were pretreated with 20 and 30 µmol/L, respectively, of JNK inhibitor SP600125 (Calbiochem-Novabiochem) for 1 hour or with 10 µmol/L p38MAPK inhibitor SB203580 (Calbiochem-Novabiochem) for 30 minutes before the NBDHEX addition and maintained throughout the experiment.
Data presentation. All of the experiments were repeated at least thrice. The data were expressed as mean ± SD, and significance was assessed by Student's t test. The criterion for statistical significance used was P < 0.05.
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The LC50 value obtained after 48 hours of treatment with each NBD derivative is reported in Table 1, showing an excellent cytotoxic activity. The LC50 values obtained on the four cell lines are comparable with the IC50 values obtained with the purified enzymatic isoform GSTP1-1.5 To support this correspondence, the compound 6-(7-nitro-2,1,3-benzoxadiazol-4-ylamino)hexanol (compound 5 of Table 1), was tested on the various cell lines as negative control. This NBD derivative (structurally similar to NBDHEX but with the sulfur atom replaced by an amine group) does not inhibit the activity of GSTP1-1 isoform. In all the tested cancer cell lines, 50 µmol/L of 6-(7-nitro-2,1,3-benzoxadiazol-4-ylamino)hexanol, managed to easily cross the membrane; however, after 48 hours of incubation, all cancer cell lines were still viable (see Table 1). The relation between the cytotoxicity of the NBD derivatives and the GST present in the cells is further confirmed by the data obtained with the hepatic carcinoma HepG2 which seemed the most resistant to treatment with the NBD derivatives. These cells express a different GST isoenzymatic pattern compared with that of most cancer cell lines (6, 28), and the lowest GST-specific activity among the cells used in our cytotoxicity trial, as shown in Table 2.
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Test of acute toxicity in vivo. To assess the acute toxicity of NBDHEX, males of the BDF1 mouse strain were treated with a single administration, through i.p. injection, with NBDHEX. The mice were followed up for 15 days and recorded for weight trend. Figure 1A, shows that, even after 15 days of treatment, the mice presented only slight weight variations. Moreover, each mouse's liver and spleen showed a weight within the norm and did not present postmortem abnormalities.
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Interestingly, not only any cytotoxic effect was observed on WBC as shown by cell count, but a slight increase of white cells, in particular neutrophils, was observed after treatment with NBDHEX (see Fig. 1C), suggesting the absence of noxious effects of NBDHEX even at the maximum concentration used (125 mg/kg).
6-(7-Nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol–induced apoptosis in K562 and CCRF-CEM cell lines. The mechanism of cell death was investigated in the human T-lymphoblastic leukemia CCRF-CEM and in the human chronic myelogenous leukemia K562 cell lines. The CCRF-CEM cells show the highest sensitivity towards the tested compounds, whereas K562 cells are known to be highly resistant to the induction of apoptosis by various anticancer agents (29). Evidences of apoptosis induction in K562 and CCRF-CEM cell lines by NBDHEX were obtained by measuring caspase 3 activation and by morphologic and flow cytometric analyzes.
Morphologic data were obtained by using the fluorescence microscope after nuclear staining with Hoechst 33342. Figure 2A shows that 24 hours of treatment of CCRF-CEM and K562 cells with 2 and 10 µmol/L NBDHEX, respectively, induces chromatin condensation and nuclear fragmentation in small rounded bodies which represent the final steps of apoptosis (30).
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To further confirm previous results, we did cytofluorimetric analysis of cells after simultaneous staining with Annexin V-FITC and PI. After 12 hours of exposure to NBDHEX, flow cytometric analysis on K562 and CCRF-CEM cell lines showed apoptosis in about 30% of total population. At 24 hours, apoptosis further increases to a value of about 45% in both cell lines (Fig. 2C). The apoptotic index perfectly coincides with the morphologic and caspase activity measurements; as expected, Annexin V values are lower because the apoptotic cells in secondary necrosis were not considered. Overall, the above results clearly indicated that the NBDHEX induced cell death by apoptosis in K562 and CCRF-CEM cells and, in our experimental conditions (NBDHEX used at a concentration five times the LC50 value of each cell line), the time course of this process was comparable in both cell lines.
Activation of the c-jun NH2-terminal kinase/c-Jun pathway. It is known that GSTP1-1 stabilizes the inactive form of JNK by a direct interaction with this mitogen-activated protein kinase (MAPK; ref. 9). Specific inhibitors of the GSTP1-1 may induce dissociation of the JNK-GSTP1-1 complex; this is followed by activation of JNK and phosphorylation of the downstream transcription factor c-Jun (9).
To verify whether the induction of apoptosis by NBDHEX involved activation of the JNK pathway, we did Western blot analyzes of both CCRF-CEM and K562 cells after treatment with NBDHEX (Fig. 3). In both cell lines, the active phospho-JNK form was evident after 30 minutes of incubation with NBDHEX and it time-dependently increased up to 24 hours. Conversely, the phosphoactivation trend of c-Jun in CCRF-CEM cell lines showed a rapid increase after 30 minutes on NBDHEX addition, although it seemed to decrease rapidly after 3 hours of treatment, reaching the basal level after 12 to 24 hours (Fig. 3A). On the other hand, phosphoactivation of c-Jun in K562 cell lines parallels the activation of JNK; phospho-c-Jun was rapidly detected after 30 minutes of incubation with NBDHEX and it remained at high levels up to 12 hours (Fig. 3B). These data suggest that apoptosis induction was strictly associated with the activation of the JNK/c-Jun pathway even if the modality of cell response seemed different. In particular, a histotype-dependent induction of the MAPK pathway could be hypothesized.
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The involvement of the JNK/c-Jun pathway in the NBDHEX-mediated apoptosis was confirmed by the experiments carried out with the specific JNK inhibitor SP600125 (31). SP600125 (30 µmol/L) suppressed after 24 hours about 70% of the apoptosis in K562 cells treated with NBDHEX. Unfortunately, in CCRF-CEM, high SP600125 concentrations induced not negligible apoptosis in the control cells. SP600125 (20 µmol/L) suppressed >50% of apoptosis after 24 hours with 13% of apoptosis in the control cells (Fig. 3D). By subtracting the contribution of apoptosis due to 20 µmol/L SP600125 alone, a net protection of about 80% can be calculated in the NBDHEX-treated cells after 24 hours. In addition, analyzing the data obtained at variable JNK inhibitor concentrations from 5 to 20 µmol/L on NBDHEX-treated CCRF-CEM, a theoretical protection from apoptosis higher than 90% can be extrapolated, at 30 µmol/L SP600125 (data not shown).
Reactive oxygen species level and redox status in cells. As previously reported, JNK dissociation from the inhibitor GSTP1-1 can be mediated by an altered intracellular redox state, in particular by ROS production (26). In fact, cytotoxic agents may generate ROS in excess of levels of the thiol buffer, and through the stress kinase cascade, they can lead to the activation of apoptotic effector molecules. We investigated if NBDHEX was able to directly or indirectly catalyze ROS formation and the role of the ROS as potential mediators of the NBDHEX-induced apoptosis. In both K562 and CCRF-CEM cell lines, incubation with NBDHEX produced an increase of ROS with a peak value at about 20 minutes (data not shown). Preincubation of CCRF-CEM cell line with 2 mmol/L of the ROS scavenger TROLOX, resulted in a significant decrement of ROS (about 60%) in the NBDHEX-treated samples (Fig. 4A). However, the decrease of ROS did not affect the extent and the time course of apoptosis, suggesting that oxidative stress is not an essential event in the apoptotic response induced by NBDHEX (Fig. 4B). Conversely, in K562 cells, the extent of apoptosis did not overlap that obtained in the absence of TROLOX. TROLOX (2 mmol/L) decreased the amount of the ROS by about 40% (Fig. 4C), and the amount of apoptotic cells proportionally decreased by about 30% after 24 hours of NBDHEX treatment (Fig. 4D), implying an additional role for ROS production in the NBDHEX-mediated apoptosis observed in K562 cells.
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-glutamyl-cysteinyl-synthetase and -glutamyltranspeptidase) has been evaluated. -Glutamyl-cysteinyl-synthetase activity was determined on K562 cells as described in Materials and Methods. Cells had a specific activity of about 1 unit/mg of protein that was not affected by the NBDHEX treatment. Moreover, NBDHEX (up to 20 µmol/L) did not affect the activity of purified -glutamyltranspeptidase (data not shown). Activation of the p38 pathway. To better clarify the mechanism of K562 cell death, we used a specific inhibitor of protein synthesis. Pretreatment of K562 with 10 µg/mL of cycloeximide strongly prevented the NBDHEX-induced apoptosis (data not shown), suggesting the involvement of transcription factors in K562 cell death. A central role in the transcription factors regulation is played by MAPKs and among them p38MAPK senses very low changes of the intracellular redox state and may be activated by alteration of the GSH/GSSG ratio (32). Thus, we studied the effect of NBDHEX on p38MAPK activation. Western blot analysis clearly shows the active form of p38 in the K562-treated cells (Fig. 6A), whereas NBDHEX failed to induce p38 phosphorilation in K562 cells pretreated with SB203580, the highly specific inhibitor of p38MAPK (ref. 33; Fig. 6B). Conversely, the phospho-p38 is mainly undetectable in CCRF-CEM cells after 24 hours of NBDHEX treatment (Fig. 6A).
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Overall, these data indicate that apoptosis stimulated by NBDHEX mainly involves the JNK pathway. In addition, only in K562 cells, the imbalance of the intracellular redox state may cause a partial involvement of the p38MAPK pathway. We cannot exclude a direct activation of p38MAPK by NBDHEX. However, this possibility seems unlikely because it does not occur in CCRF-CEM cells.
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Discussion |
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-complex strongly stabilized by both G-site and H-site. Thus, the -complex may induce conformational changes in both these two subsites. The -complex, once in solution, easily dissociates (KD about 10–2 mol/L) into NBDHEX and GSH, and this instability is a fortunate event because it avoids the excretion of the -complex from the cell through specific membrane pumps (multidrug resistance protein; ref. 35).
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We note that in both CCRF-CEM and K562 cell lines, incubation with NBDHEX produced a small increase of ROS concentration. Anyway, apoptosis seems completely ROS-independent in CCRF-CEM cells, whereas in K562 cells, the effect of TROLOX indicated that 30% of apoptosis is mediated by ROS (see Fig. 4). Moreover, the experiments done in the presence of SB203580 suggest that p38MAPK was involved in the death process only in K562 cells (see Fig. 6) where we observed a concomitant alteration of the GSH/GSSG ratio which may reflect a different sensitivity of K562 cells to ROS insult. Overall, the results indicate that even in K562 cells the dissociation of the JNK-GSTP1-1 complex remains the main pathway of the NBDHEX-triggered apoptosis. The dissociation of the JNK-GSTP1-1 complex is not induced by oxidative stress but is a direct consequence of the interaction between NBDHEX and the JNK-linked GSTP1-1. Genesis of ROS in NBDHEX-treated cells and the very different response to this stress condition of K562 and CCRF-CEM cells are unclear at the present and work is in progress to clarify these aspects.
We must underline that NBDHEX is able to induce apoptosis in the chronic myelogenous leukemia (K562) cells in spite of the presence of the apoptotic suppressor Bcr-Abl (36). The persistent activation of JNK and c-Jun observed in these cells is peculiar given the high resistance of K562 cells to the induction of apoptosis by various agents including camptothecin, 1-ß-D-arabinofuranosylcytosine, etoposide, paclitaxel, staurosporine, and anti-Fas antibodies (37).
Overall the findings of this study disclose interesting perspectives of the novel NBD derivatives in the treatment of leukemia and related disorders. In the past, traditional GST inhibitors have been mainly used to prevent the multidrug resistance phenomenon in patients undergoing treatment with other chemotherapeutic agents. The NBD derivatives, besides their possible utilization in association with other chemotherapeutic agents, are themselves toxic for tumor cells. Furthermore, the tumor cells may represent the primary target of these molecules because most of these cells overexpress GSTP1-1 and this enzyme binds NBD derivatives with high affinity. The low toxicity observed in vivo (as deduced by the good recovery of treated mice, by the absence of histologic abnormality on liver and spleen tissues, and by the absence of cytotoxic effects on WBC and RBC) seems to confirm this idea, indicating a promising possible use of these compounds as anticancer agents. Work is in progress in our laboratory to address whether NBDHEX shows selective antitumor activity in suitable in vivo model systems.
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Acknowledgments |
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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.
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Footnotes |
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5 G. Ricci et al. 7-Nitro-2,1,3-benzoxadiazole derivatives: a new class of suicide inhibitors for glutathione S-transferases, submitted for publication. 
Received 10/29/04. Revised 1/24/05. Accepted 2/24/05.
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-Glutamyl transpeptidase. Methods Enzymol 1981;77:237–53.[Medline]
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G. Ricci, F. De Maria, G. Antonini, P. Turella, A. Bullo, L. Stella, G. Filomeni, G. Federici, and A. M. Caccuri 7-Nitro-2,1,3-benzoxadiazole Derivatives, a New Class of Suicide Inhibitors for Glutathione S-Transferases: MECHANISM OF ACTION OF POTENTIAL ANTICANCER DRUGS J. Biol. Chem., July 15, 2005; 280(28): 26397 - 26405. [Abstract] [Full Text] [PDF]
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) 125, (
) 25, (
) and 5 mg/kg. The remaining two groups were used as controls and treated with the following administrations: (
) an olive oil vehicle containing 2.5% DMSO and (
) a physiological solution. Weight of each mouse was monitored for 15 days showing only slight weight variations. B, liver and spleen tissues, obtained after 15 days from the NBDHEX treatment, were stained with H&E. Any toxic effect due to the NBDHEX administration was detected. Liver tissues of mice treated with 125 mg/kg NBDHEX (a and b), 5 mg/kg NBDHEX (c), oil vehicle containing 2.5% DMSO (d), oil vehicle (e), and physiological solution (f). Spleen tissues of mice treated with 125 mg/kg NBDHEX (a and b), 5 mg/kg NBDHEX (c), oil vehicle containing 2.5% DMSO (d), oil vehicle (e), and physiological solution (f). a, magnification 10x; b-f, magnification 4x. C, hematologic variables were analyzed in mice after 15 days from the NBDHEX treatment. Absolute number of RBC (a) and hematocrit (b). WBCs are expressed as number of cells per µL (c); percentage of neutrophils (d) and lymphocytes (e). Columns, mean; bars, ±SD.
F), measured within 1 minute from the substrate addition, normalized per 106 cells. NBDHEX treated cells (
