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Vol. 2, 409-417, April 2003     Molecular Cancer Therapeutics
© 2003 American Association for Cancer Research

JS-K, a Glutathione/Glutathione S-Transferase-activated Nitric Oxide Donor of the Diazeniumdiolate Class with Potent Antineoplastic Activity1

Paul J. Shami2, Joseph E. Saavedra, Lai Y. Wang, Challice L. Bonifant, Bhalchandra A. Diwan, Shivendra V. Singh, Yijun Gu, Stephen D. Fox, Gregory S. Buzard, Michael L. Citro, David J. Waterhouse, Keith M. Davies, Xinhua Ji and Larry K. Keefer

Division of Medical Oncology, Department of Internal Medicine, University of Utah and Salt Lake City Veterans’ Administration Medical Centers, Salt Lake City, Utah 84148 [P. J. S., L. Y. W.]; Basic Research Program [J. E. S., B. A. D., G. S. B., M. L. C.] and Analytical Chemistry Laboratory [S. D. F.], Science Applications International Corporation-Frederick, National Cancer Institute at Frederick, Frederick, Maryland 21702; Department of Pharmacology, University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania 15213 [S. V. S., Y. G.]; Macromolecular Crystallography Laboratory, National Cancer Institute at Frederick, Frederick, Maryland 21702 [Y. G., X. J.]; Department of Chemistry, George Mason University, Fairfax, Virginia 22030 [K. M. D.]; and Laboratory of Comparative Carcinogenesis, National Cancer Institute at Frederick, Frederick, Maryland 21702 [C. L. B., D. J. W., L. K. K.]

2 To whom requests for reprints should be addressed, at SLC VA Medical Center, Box 151M, 500 Foothill Boulevard, Salt Lake City, UT 84148. Phone: (801) 582-1565; Fax: (801) 583-9624; E-mail: p.shami{at}m.cc.utah.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have previously shown that nitric oxide (NO) inhibits growth and induces differentiation and apoptosis in acute myeloid leukemia cells, with the HL-60 human myeloid leukemia line being particularly sensitive to NO-mediated cytolysis. With the goal of identifying a prodrug that can target NO to the leukemia cells without inducing NO-mediated systemic hypotension, we have screened a series of O2-aryl diazeniumdiolates designed to be stable at physiological pH but to release NO upon reaction with glutathione. O2-(2,4-Dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K) proved to be the most active antiproliferative agent among those tested in HL-60 cells, with an IC50 of 0.2–0.5 µM. After 5 days of exposure to 0.5 µM JS-K, HL-60 cells had differentiated and acquired some of the phenotypic features of normal monocytes. One- to 2-day treatment with JS-K at concentrations of 0.5–1 µM resulted in apoptosis induction in a concentration- and caspase-dependent manner. JS-K also inhibited the growth of solid tumor cell lines but to a lesser extent than HL-60 cells. JS-K was administered i.v. to nonobese diabetic-severe combined immune deficient mice at doses of up to 4 µmol/kg without inducing significant hypotension. The growth of s.c. implanted HL-60 cells was reduced by ~50% when the mice received i.v. injections three times/week with 4 µmol/kg boluses of JS-K. Histological examination of tumor explants from JS-K-treated animals revealed extensive necrosis. Similar results were seen with s.c. human prostate cancer (PPC-1) xenografts. Our data indicate that JS-K is a promising lead compound for the possible development of a novel class of antineoplastic agents.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
AML3 is a life-threatening disease with an annual incidence of 2.25 per 100,000 (1). Despite therapy with different classes of chemotherapeutic agents, including anthracyclines, 1-ß-D-arabinofuranosylcytosine, etoposide, all-trans-retinoic acid, as well as high-dose therapy with stem cell rescue, the overall 5-year disease-free survival remains ~30%. This illustrates the need for agents with novel mechanisms of action.

This study is based on the premise that the unusual sensitivity of AML cells to the cytotoxic effects of NO can be exploited for improved therapy of this malignancy. We previously showed that NO-releasing drugs of the diazeniumdiolate class induce apoptosis in the HL-60 human myeloid leukemia cell line at relatively low concentrations (for example, an IC50 of 0.006 mM for a cell-permeant prodrug that is activated for NO release by intracellular esterases; Ref. 2). This is in marked contrast to, for example, normal vascular smooth muscle cells, which experience cytostasis without toxicity in the continuous presence of 0.2–0.5 mM (Z)-1-[2-(2-Aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (half-life for NO release of 20 h) for 6 days (3), and to primary hepatocytes, which can actually be protected from apoptosis by exposure to a diazeniumdiolate that is activated for NO release by cytochrome P450 (4).

One might, therefore, expect NO-based therapies to exhibit some selectivity for the NO-sensitive leukemia cells relative to normal tissues. With this in mind, we have screened a library of arylated diazeniumdiolates that were designed to be activated for NO release by reaction with cellular thiols such as GSH (5) with or without catalysis by GST, the major isoforms ({alpha}, µ, {pi}) of which are expressed in 75, 55, and 95% of AML cases, respectively (6). We report here on the in vitro and in vivo antineoplastic activity of the most potent growth inhibitor of this family tested to date, JS-K (structure in Fig. 1).



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  		Fig. 1. Structures of JS-K, our control arylating agent CDNB, and their common GSH conjugate (DNP-SG). The structures of the Meisenheimer complexes presumed to be intermediates in the two conjugation reactions are also shown. 4-Carbethoxy-PIPERAZI/NO (the coproduct of the GSH/JS-K reaction) generates NO spontaneously at physiological pH as shown; its half-life for NO release at 37°C in 0.1 M phosphate buffer (pH 7.4) was found to be 6.0 min (k = 1.9 x 10-3 s-1).

 

   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals.
JS-K (7) and 4-carbethoxy-PIPERAZI/NO (Ref. 8; structures shown in (Fig. 1) were synthesized as described previously. S-(2,4-Dinitrophenyl)glutathione was prepared by the method of Mancini et al. (9). The pan-caspase inhibitor Z-VAD-FMK was from Biomol (Plymouth Meeting, PA). Daunorubicin and etoposide were from Dr. Grayden Harker (University of Utah). HPLC grade solvents were purchased from VWR Scientific Co. (South Plainfield, NJ). All other chemicals were from Sigma (St. Louis, MO) unless otherwise noted.

Chromatography and MS.
HPLC separations were carried out with a Phenomenex Luna 5 µ C18(2) column (Torrance, CA) using a gradient of acetonitrile:water (each containing 0.1% formic acid) at a flow rate of 1 ml/min (percentage of acetonitrile: 25% for 0–5 min followed by a linear program to 70% at 15 min).

HPLC-MS studies were performed on an Agilent Capillary Series 1100 LC/MSD Ion Trap mass spectrometer with electrospray ionization in the positive ion mode. Separations were effected as above, except that the flow rate was 15 µl/min, and the gradient was 10% acetonitrile for 0–5 min followed by a linear program to 70% at 15 min.

Molecular Modeling.
CDNB is a model substrate used extensively for monitoring activity of GST (10). It has been shown that GSTs with a higher specific activity toward CDNB stabilize the Meisenheimer complex (Fig. 1) at the transition state better than the isoforms with poor catalytic activity for CDNB-GSH conjugation (5). Crystal structures of a transition state analogue, GSTCD-, in complex with rGSTM1-1, a µ class rat GST isoform (11), and with hGSTP1-1 (isoleucine-104, alanine-113 variant), a {pi} class human GST isoform (12), have been reported and provided the foundation for modeling the transition state of GST-catalyzed GSH conjugation of JS-K. The initial model of the Meisenheimer complex of GSH and JS-K for hGSTM1-1 and that for hGSTP1-1 built based on the crystal structures of GSTCD- in rGSTM1-1 (11) and that in hGSTP1-1 (12), respectively. The models were subject to geometry optimization using the conjugate gradient method of Powell (13) and docked into the active sites of ligand-free hGSTM1-1 and hGSTP1-1 built based on the rGSTM1-1 (11) and hGSTP1-1 (12) structures, respectively. The geometry of the protein-Meisenheimer complexes was then optimized, and the energy was minimized (13). Both complexes were built in dimeric form considering the fact that the biologically active forms of GSTs are dimeric proteins and that the glutathionyl moiety of GSH interacts with the side chains from both subunits (14–17). The Engh and Huber (18) geometric parameters were used as the basis of the force field. No crystal structure of {alpha} class GST-bound GSTCD- is available. The Meisenheimer complex for {alpha} class GST was therefore built by modifying those for rGSTM1-1 and hGSTP1-1 based on the structures of hGSTA1-1-bound S-benzyl-GSH (19) and the GSH conjugate of ethacrynic acid (20). After energy minimization, the Meisenheimer complex for hGSTA1-1 was docked in the active center of the enzyme, and the protein-Meisenheimer complex was subject to energy minimization as described above. The molecular modeling studies were carried out with program suites O (21) and X-PLOR (22).

Determination of Specific Activity of Human GSTs toward JS-K.
Purified preparations of recombinant hGSTA1-1, hGSTM1-1, and hGSTP1-1 were obtained from Panvera (Madison, WI). The activity of human GST toward CDNB was determined, as described by Habig et al. (10), before activity measurements with JS-K to ensure that the enzyme preparations were catalytically active. For activity measurement toward JS-K, the reaction mixture in a final volume of 1 ml contained 100 mM potassium phosphate buffer (pH 6.5), 1 mM GSH, 0.045 mM JS-K, and an appropriate amount of human GST isoenzyme protein. The reaction was started by the addition of JS-K, and the rate of reaction was monitored by measuring decrease in absorbance of JS-K at 298 nm because of its reaction with GSH. The specific activity toward JS-K was calculated using an extinction coefficient of 18 mM-1cm-1 at 298 nm.

Measurement of NO Release.
Chemiluminescence detection and quantification of NO evolving from the reactions of JS-K were conducted using an NO-specific Thermal Energy Analyzer (Model 502A; Thermedics, Analytical Instrument Division, Waltham, MA) essentially as described previously (23). Briefly, pH 7.4 phosphate buffer containing 1 mM GSH was sparged with inert gas until a steady detector response was established. Where indicated, GSTs were added to a final concentration of 1.67 µg of enzyme/ml. The NO release profile was followed at 37°C for 45 min after injecting JS-K at a final concentration of 133 nM to start the reaction. The resulting curve was integrated to quantify the amount of NO released/mol of compound.

Cell Lines and Culture Conditions.
HL-60, DLD1, and U937 cells were from American Type Culture Collection (Manassas, VA). Meth A cells were from Dr. Wolfram Samlowski (University of Utah). The PPC-1 cell line was provided by Dr. Graeme Bolger (University of Alabama). For the cell growth and apoptosis experiments, cells were cultured at a density of 150,000 cells/ml in RPMI 1640 with 10% fetal bovine serum at 37°C in a 5% CO2-humidified atmosphere. Agents were added at the indicated concentrations 24 h after culture initiation. At the indicated time intervals, cells were harvested and washed twice in PBS before processing for analysis of growth, differentiation, and apoptosis.

Cell Growth, Differentiation, and Apoptosis Assays.
The number of viable cells was determined using the MTT assay according to the manufacturer’s protocol (Promega, Madison, WI) or using a Coulter counter. Differentiation was evaluated using Wright and NSE staining of cells collected on microscope slides by cytospin as described previously (24). Apoptosis was assayed by flow cytometry and by determining DNA fragmentation using agarose gel electrophoresis as described previously (25). For the flow cytometry assay, we used the propidium iodide staining method of Nicoletti et al. (26).

In Vivo Studies of JS-K.
NOD/SCID mice were bred and maintained at the Huntsman Cancer Institute at the University of Utah. Experiments were performed on male or female mice 6–8 weeks of age at the Animal Care Facility of the Salt Lake City Veterans’ Administration Medical Center after approval by the Institutional Animal Care and Use Committees. We measured systolic blood pressure on unanesthetized NOD/SCID mice using an occluding tail cuff and a pulse transducer connected to a blood pressure transducer/monitor from World Precision Instruments (Sarasota, FL). Signals from the blood pressure monitor and pulse transducer were transmitted to a MacLab2 data acquisition device (purchased from Stoelting, Wood Dale, IL) that feeds directly into a Macintosh computer. The recorded data were analyzed using the Chart data analysis software purchased from Stoelting. Measurements were done in triplicate at each time point.

To study the in vivo antineoplastic potency of JS-K, NOD/SCID mice received injections in the flanks s.c. with HL-60 or PPC-1 cells (2.5 x 106 cells/flank). When s.c. tumors were palpable, treatment with JS-K or an equal volume of vehicle (20% DMSO in PBS) was started using the indicated doses and route. Tumor size was measured daily or every other day using a Vernier caliper. Tumor volume was calculated using the formula: width x length x [(width + length)/2] x 0.5236. Fifteen to 20 days after tumor cell implantation, animals were sacrificed by CO2 inhalation, and tumors were collected for histochemical analysis.

Histological Analysis of Tumors.
At the completion of the experiments, animals were sacrificed, and s.c. tumors were dissected out, fixed in 10% formaldehyde, and imbedded in paraffin. Four-µm sections were cut and stained with H&E.

Calculations and Statistical Analysis.
Results are expressed as averages of multiple experiments with SE. SE was calculated as the SD of different measurements divided by the square root of the number of measurements. Differences were considered statistically significant if the P was < 0.05 as calculated using the t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Reactivity of JS-K with GSH.
The diazeniumdiolate ion has been judged to resemble chloride as a leaving group in SNAr reactions (7). Because CDNB (structure shown in (Fig. 1) is known to react with GSH, we anticipated that JS-K would be similarly converted to DNP-SG (structure also shown in (Fig. 1). This was confirmed by HPLC-MS; as shown in Fig. 2A, an 85% conversion of JS-K to DNP-SG occurred within a 30-min incubation period at 37°C in pH 7.4 phosphate buffer. Similar results were seen in RPMI 1640 cell culture. Pseudo-first-order kinetic plots for the reaction of GSH with JS-K in 0.1 M phosphate buffer (pH 7.4) were obtained with GSH (1–5 mM) in large excess of the substrate. Excellent first-order behavior was observed over several half-lives and measured first-order rate constants showed a linear dependence on (GSH; Fig. 2C). The slope and y intercept of the line yielded values for the second-order [k2 = (1.02 + 0.04) M-1 s-1] and first-order [k1 = (4 + 12) x 10-5 s-1] rate constants for the reactions of JS-K with GSH and water, respectively, at 37°C in 0.1 M phosphate buffer, pH 7.4. The UV spectral changes accompanying the reaction in a second cell culture medium (DMEM) are shown in Fig. 2B.



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  		Fig. 2. Reactions of JS-K with thiols. A, HPLC traces of a reaction mixture containing 50 µM JS-K and 500 µM GSH at 37°C in 0.1 M phosphate (pH 7.4) at an observing wavelength of 300 nm immediately after mixing (top) and 30 min later (bottom). The structures of JS-K and its GSH conjugate DNP-SG (extinction coefficients at 300 nm of 16 and 3.7 mM-1 cm-1, respectively) are shown in Fig. 1; 2,4-DNP is the JS-K hydrolysis product, 2,4-dinitrophenol, and DMSO is the cosolvent. B, UV spectral changes during 30 min at 37°C of a 50 µM JS-K solution in serum-free DMEM in the absence (left) and presence (right) of 1 mM GSH. Note that in this cystine-containing medium, the product is S-(2,4-dinitrophenyl)cysteine, {lambda}max 354 nm, rather than DNP-SG, {lambda}max 340 nm. C, increases in JS-K consumption rate at an initial concentration of 50 µM in 0.1 M phosphate (pH 7.4) at 37°C as a function of increasing GSH concentration.

 
Hydrolysis of JS-K.
JS-K proved resistant to simple hydrolysis under these conditions, as reflected in the near-zero y intercept of Fig. 2C. The value of k1 obtained from the intercept is in statistical agreement with the rate constant for JS-K hydrolysis (1 x 10-6 s-1) measured separately in the absence of GSH. The small amount of the hydrolysis product (2,4-dinitrophenol) seen in the chromatogram of Fig. 2A was apparently formed in the DMSO stock solution, which in this case had been stored in the refrigerator with intermittent use during several weeks. Hydrolysis was much more facile at pH 12, which was expected for a compound type designed to be activated for NO release by nucleophilic attack.

Solubility Limits.
JS-K showed an interesting but potentially nettlesome tendency to remain supersaturated in aqueous solutions, only to separate from the aqueous phase at a time course that was difficult to predict or control. For example, our first attempts to follow the hydrolysis of 50 µM JS-K spectrophotometrically sometimes showed little change, as expected from Fig. 2C, but at other times revealed a variable and sometimes rapid rate of absorbance loss without production of new maxima. The problem could be overcome by cleaning the cuvette with nitric acid before filling it with buffer then adding the DMSO/JS-K stock solution, in which case absorbance changed little with time. However, rinsing this solution out with distilled water followed by refilling the cuvette with buffer and adding the stock solution often led to shrinking absorbance with concomitant appearance of cloudiness in the cuvette; dissolution of the insoluble material, after isolating it by centrifugation, showed that the resulting solid (dissolved in acetonitrile) was essentially pure JS-K in an amount equivalent to the quantity lost from the supernatant. Quantification of the JS-K in the buffer by HPLC and UV spectrophotometry after all spectral changes had stopped indicated that JS-K’s solubility limit in 0.1 M phosphate buffer at 37°C and pH 7.4 containing 1% DMSO was ~10 µM. The relative insolubility of JS-K in aqueous media should be taken into account during any experimental work involving dilution of organic JS-K solutions with aqueous media because the observed tendency toward supersaturation can lead to JS-K concentrations that are much lower or higher than expected and thus to erroneous results.

Catalysis of the GSH/JS-K Reaction and NO Release by GST.
The reaction of GSH with CDNB (Fig. 1) is catalyzed by several classes of human GSTs, and, thus, this electrophilic substrate is often used for quantifying their activity. Given the similarity of diazeniumdiolate ions to chloride as a leaving group in SNAr reactions (7), we expected JS-K also to undergo GST-catalyzed conjugation with GSH. To gain insights into the effect the obvious steric differences between chloride and the diazeniumdiolate ion shown in Fig. 1 may imply, we modeled the accommodation of the Meisenheimer complex of JS-K in the active sites of the three major classes of human GSTs, i.e., hGSTA1-1, hGSTM1-1, and hGSTP1-1. As illustrated in Fig. 3, A–C, both hGSTA1-1 and hGSTM1-1 classes of GSTs accommodate the Meisenheimer complex very well, but hGSTP1-1 appears to have serious steric conflicts with the diazeniumdiolate moiety of the transition state complex. On the basis of molecular modeling, we predicted that hGSTA1-1 and hGSTM1-1 should be more effective than hGSTP1-1 for catalyzing the GSH conjugation of JS-K.



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  		Fig. 3. Catalysis of JS-K’s NO release by GST. A–C illustrate the accommodation of the Meisenheimer complex of JS-K in the active center of (A) hGSTA1-1, (B) hGSTM1-1, and (C) hGSTP1-1 as predicted by the molecular modeling studies. The active centers of the GSTs are shown as surface representations and the Meisenheimer complexes as ball-and-stick models with atomic color scheme (carbon, gray; nitrogen, blue; oxygen, red; and sulfur, yellow). The illustration was prepared using Grasp (29) and Raster3D (30). D, chemiluminescence traces showing the time course of NO release from 0.67 µM JS-K at 37°C in 0.1 M phosphate (pH 7.4) containing 1.0 mM GSH both alone and with 1.67 µg/ml hGSTA1-1, hGSTM1–1, or hGSTP1–1.

 
These predictions were confirmed by determining the activities of recombinant hGSTA1-1, hGSTM1-1, and hGSTP1-1 preparations toward JS-K. The data are summarized in Table 1. Specific activities of GSTs toward CDNB were determined before activity measurement with JS-K to ensure that the enzyme preparations were catalytically active. The specific activities of the GSTs toward CDNB were comparable with the values published in the literature (Ref. 27; Table 1). In agreement with our prediction, hGSTP1-1 was much less active than hGSTA1-1 or hGSTM1-1 for GSH conjugation of JS-K.


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  		Table 1 Specific activities (µmol min-1 mg-1) of GST-catalyzed glutathionylation of CDNB and JS-K in 0.10 M phosphate buffer at pH 6.5 and 25°Ca

 
That these reactions led to NO generation as predicted was demonstrated by purging gases from the solution as they formed into an NO-specific chemiluminescence detector. The results are shown in Fig. 3D. Consistent with JS-K’s resistance to hydrolysis but its reactivity toward GSH as noted above, no NO was detected until 1 mM GSH was added to the pH 7.4 phosphate/0.67 µM JS-K solution. In the absence of enzyme, NO release began immediately upon adding GSH, increasing in rate until plateauing at 8 min, and integrating to a total of 1.1 mol of NO/mol of JS-K within 43 min of mixing. The hGSTP1-1 isoform catalyzed this reaction, but only weakly; that this was not attributable to a deleterious effect of JS-K exposure on the enzyme’s activity was demonstrated in its unfettered ability, in the presence of up to 80 µM JS-K, to catalyze CDNB’s conjugation with GSH. The hGSTA1-1 and hGSTM1-1 isoforms proved much superior to hGSTP1-1 as catalysts for JS-K conjugation. The data of Fig. 3D on NO release are thus consistent with the conclusion from the JS-K consumption studies of Table 1 that JS-K is metabolized much better by hGSTA1-1 and hGSTM1-1 than by hGSTP1-1.

Growth Inhibitory Properties of JS-K.
Fig. 4A shows a comparison between the growth inhibitory ability of JS-K and those of the chemotherapeutic agents daunorubicin and etoposide in our HL-60 assay system. The IC50s of JS-K, daunorubicin, and etoposide were 0.5, 0.01, and 0.3 µM, respectively. CDNB, a compound with the same aryl ring as JS-K that does not release NO (Fig. 1), inhibited the in vitro growth of HL-60 cells but at much higher concentrations, with an IC50 estimated at 6.7 µM (data not shown). JS-K also inhibited the growth of U937 (monocytic leukemia) cells with an IC50 of 0.3 µM (data not shown). Solid tumor cell growth was also inhibited by JS-K, although to a lesser extent than leukemia cells (Fig. 5); the IC50s for the three lines we tested, PPC-1, DLD-1, and Meth A, were an order of magnitude greater than those for the two leukemia lines.



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  		Fig. 4. Antiproliferative and proapoptotic effects of JS-K on HL-60 leukemia cells in vitro. A, comparison of the antiproliferative effects of JS-K with those of other chemotherapeutic agents. Cells were cultured with JS-K, daunorubicin, or etoposide for 3 days at the indicated concentrations. Cell viability was determined using the MTT assay (averages and SE of three separate experiments). B, apoptosis induction by JS-K. Cells were cultured with JS-K at the indicated concentrations. At 72 h, the percentage of apoptotic cells was determined by flow cytometry (see text; averages and SE of three separate experiments). C, DNA laddering in HL-60 cells resulting from 3-day exposure to 1 µM JS-K (gel representative of three different experiments; MWM, molecular weight marker). D, reversal by the caspase inhibitor C-VAD-FMK of JS-K’s cytostatic and proapoptotic effects on HL-60 cells. Cells were cultured with JS-K (0.75 µM), C-VAD-FMK (50 µM), or the combination for 3 days. Cell viability and apoptosis were determined using the MTT assay and flow cytometry, respectively (averages and SE of three separate experiments).

 


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  		Fig. 5. Inhibitory effect of JS-K on three different solid tumor cell lines. PPC-1 (prostate), DLD-1 (colon), and Meth A (mammary) cells were cultured with JS-K at the indicated concentrations for 3 days. Cell viability was determined using the MTT assay (averages and SE of three separate experiments).

 
To determine whether modulation of the GST pathway affects JS-K’s antineoplastic properties, we performed experiments using NAC or BSO. NAC increases intracellular GSH levels, whereas BSO inhibits its synthesis (28). Treatment of HL-60 cells with NAC (0.3–0.5 mM) or BSO (0.2–0.3 mM) did not significantly affect cell growth. Pretreatment of the cells for 2–6 h with NAC prevented the JS-K-induced growth inhibition. Pretreatment of HL-60 cells with BSO for 2–6 h did not prevent the JS-K-induced growth inhibition, whereas pretreatment of the cells with BSO for 24 h enhanced it (data not shown).

Induction of Leukemia Cell Apoptosis by JS-K.
Because we had previously shown that spontaneous and esterase-activated NO generators induce apoptosis in leukemia cells (2, 25), we sought to determine whether JS-K had a similar effect. Three days after addition of JS-K at concentrations of 0.5 and 1 µM, the percentage of apoptotic HL-60 cells increased from 7 to 27 and 43%, respectively (Fig. 4B). The flow cytometry experiments were confirmed with DNA laddering assays (Fig. 4C). The pan-caspase inhibitor C-VAD-FMK prevented the JS-K-induced growth inhibitory and apoptotic effects (Fig. 4D).

Effect of JS-K on Leukemia Cell Differentiation.
We have previously shown that NO induces HL-60 cells to differentiate along the monocytic phenotype (24). We therefore determined whether JS-K induced differentiation as well. HL-60 cells were treated with JS-K at a concentration of 0.5 µM for 3–5 days. Wright stain revealed morphological changes consistent with a monocytic phenotype, namely development of folded nuclei, large cytoplasms, and cytoplasmic vacuoles (Fig. 6). NSE (an enzyme specific to the monocytic lineage) staining showed that JS-K increased the percentage of HL-60 cells expressing NSE from 1 to 40% (Fig. 6).



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  		Fig. 6. Effect of JS-K on differentiation of leukemia cells. HL-60 cells were cultured with (right) and without (left) 0.5 µM JS-K for a period of 5 days. Differentiation was assessed morphologically (Wright stain, top) and by determining NSE (bottom) expression (photomicrographs of one experiment representative of three).

 
In Vivo Effects of JS-K.
Before determining in vivo antineoplastic activity of JS-K, we next sought to identify a dose that would not induce significant hypotension in NOD/SCID mice. Using i.v. administration, we were able to escalate the dose of JS-K up to 4 µmol/kg without observing significant hypotension, whereas doses of 5 µmol/kg or higher had a notable hypotensive effect (data not shown). Using estimates of the mouse blood volume, 4 µmol/kg of JS-K would be expected to yield peak blood levels of ~17 µM, which is far above its in vitro IC50.

NOD/SCID mice were then implanted s.c. in the flanks with 2.5 x 106 HL-60 cells/flank. When the tumors were palpable, treatment was started with JS-K administered i.v. via the tail vein three times/week at a dose of 4 µmol/kg. Control mice received an equal volume of vehicle through the same route and according to the same schedule. JS-K treatment induced a significant inhibition of in vivo leukemia cell growth (Fig. 7A). Sixteen days after starting therapy, the average tumor volumes in control and JS-K-treated mice were 8.34 + 0.72 and 3.13 + 1.14 cm3 (P = 0.039), respectively, reflecting a >50% reduction in tumor volume in treated mice. Histological analysis of HL-60 cell tumors obtained from vehicle-treated mice revealed a uniform population of densely packed myeloblasts. The cells were highly invasive, penetrating the surrounding tissues, and showing high mitotic activity. Necrosis was minimal. On the other hand, histological analysis revealed extensive (>50%) cell necrosis in HL-60 cell tumors obtained from the JS-K-treated mice as compared with ~10% in controls (Fig. 7B).



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  		Fig. 7. Effects of i.v. JS-K on growth of both human leukemia and solid tumor xenografts in NOD-SCID mice. A, growth rate of tumors produced when 2.5 x 106 HL-60 cells were implanted s.c. in NOD/SCID mice [3 animals each for JS-K and vehicle treatments]. When tumors became palpable, JS-K was administered i.v. at a dose of 4 µmol/kg three times/week. Tumor volumes were measured at regular intervals. At the time of experiment termination, tumor implants were dissected out and analyzed histologically. Differences between vehicle and JS-K values from days 9–16 were significant at the P = 0.05 level. B, JS-K induced extensive necrosis in these tumors (photomicrograph from 1 animal representative of 3; H&E stain, x100 magnification). C, growth rate of tumors produced when 2.5 x 106 human prostate carcinoma (PPC-1) cells were implanted s.c. in NOD/SCID mice (5 animals each for JS-K and vehicle treatments) and followed as in A above. Differences between vehicle and JS-K values were significant at the P = 0.05 level for all time points after day 1. D, JS-K induced extensive necrosis in these tumors (photomicrograph from 1 animal representative of 5; H&E stain, x100 magnification).

 
To determine whether JS-K inhibits the in vivo growth of solid tumor cells, NOD/SCID mice were implanted with 2.5 x 106 PPC-1 (prostate carcinoma) cells and treated with 4 µmol/kg JS-K or an equal volume of vehicle i.v. three times/week. Similar to the observation with HL-60 cells, JS-K treatment inhibited the growth of PPC-1 cells in vivo. Nineteen days after start of therapy, s.c. tumor implant volumes were 0.368 + 0.082 and 0.107 + 0.053 cm3 (P = 0.0073) in vehicle and JS-K-treated animals, respectively (Fig. 7C). Similar to HL-60 cells, PPC-1 cells were highly aggressive and invaded the surrounding tissues. Histological analysis revealed extensive tumor necrosis in implants obtained from JS-K-treated animals (Fig. 7D).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results suggest that JS-K might serve as a promising lead compound in the search for new classes of antineoplastic agents with novel mechanisms of action. In a test of its antiproliferative effect on the HL-60 human myeloid leukemia cell line, its consistently submicromolar IC50 (three separate determinations of 0.2, 0.5, and 0.5 µM) compared favorably with those of the clinical antileukemics daunorubicin (0.01 µM) and etoposide (0.3 µM). Chemical characterization of JS-K showed that it resists hydrolysis in the absence of strong nucleophiles but generates copious NO upon reaction with GSH. The experiments additionally confirmed predictions based on molecular models of transition state geometry that two of three major human isoforms of GST could strongly catalyze NO release from JS-K.

Consistent with our hypothesis that such an NO prodrug might direct its toxic action selectively to the NO-sensitive malignant cells while sparing less sensitive normal tissues, JS-K injected i.v. three times/week inhibited the growth of s.c. tumors formed from HL-60 xenografts in NOD-SCID mice by approximately one-half. This suggests that JS-K may serve as a useful lead for developing therapies against leukemia.

As far as mechanism of action is concerned, the in vitro experiments showed all of the characteristics of previous studies with structurally different NO donors (including induction of both apoptosis and differentiation in HL-60 cells) and were thus consistent with NO-induced cytolysis. However, it is important to recognize that other pathways may also be operative. For example, arylation of thiol groups in critical protein residues could be contributing to the toxicity; however, this may be a minor effect, as CDNB (which transfers the same 2,4-dinitrophenyl group to cellular nucleophiles as JS-K but without generating any NO) was an order of magnitude less potent in the HL-60 screen. Whether there is synergy between the growth inhibitory effects of NO and arylation remains to be determined. Transcarbamoylation is also a possible route to toxicity. The finding that N-acetyl-L-cysteine prevented the growth inhibitory effects of JS-K while BSO enhanced them suggests that one or more reactive nitrogen/oxygen species (NO and/or the product of NO’s reaction with other reactive oxygen species) play a key role in effecting the antineoplastic activity of JS-K.

As to the specific molecular targets that may be involved, exposure to JS-K can in principle render any critical thiol group nucleophilically unreactive by S-nitrosation or S-(2,4-dinitrophenyl)ation. It is likely that the cysteine-containing caspases are directly or indirectly among the mechanistically important targets of JS-K’s action because the nonspecific caspase inhibitor C-VAD-FMK reduced JS-K’s cytostatic and apoptotic effects on HL-60 cells. Other pathways are possible, though, and a fuller explanation of the drug’s antineoplastic activity must await the outcome of future hypothesis testing.

Whatever the mechanism(s) that may be involved, the present results support the choice of JS-K as a worthy lead compound for additional drug discovery efforts. The significant in vivo activity demonstrated in Fig. 7 was seen in first generation experiments that could hardly be viewed as optimized; the dose regimen was chosen based on preliminary studies of bolus sizes that could be administered without inducing systemic hypotension or other manifestations of toxicity. It is entirely possible that a continuous administration schedule would be much more effective. Structural modification aimed at improving the problematic solubility of this compound could beneficially affect its absorption, distribution, and transport properties. With substantial efforts underway in other laboratories4 as well as our own aimed at further characterizing and refining the chemotherapeutic potential of this interesting lead, we are hopeful that our ultimate goal of introducing new and improved treatments for AML and other malignant diseases will be significantly advanced.


   Footnotes
 
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 indi-cate this fact.

1 This work is supported by a Translational Research Award from the Leukemia and Lymphoma Society (to P. J. S.). This project was also supported, in part, through National Cancer Institute Contract No. NO1-CO-12400 (J. E. S., B. A. D., S. D. F., G. S. B., M. L. C.), and National Institute of Environmental Health Sciences Grant ES09140 (to S. V. S.). Back

3 The abbreviations used are: AML, acute myeloid leukemia; BSO, buthionine sulfoximine; CDNB, 1-chloro-2,4-dinitrobenzene; DNP-SG, S-(2,4-dinitrophenyl)glutathione; GSH, glutathione; GST, glutathione S-transferase; GSTCD-, 1-(S-glutathionyl)-2,4,6-trinitrocyclohexadienate anion; HPLC, high performance liquid chromatography; JS-K, O2-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate; MS, mass spectrometry; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NAC, N-acetyl-L-cysteine; NO, nitric oxide; NOD-SCID, nonobese diabetic-severe combined immune deficient; NSE, nonspecific esterase; xGSTY1-1, class Y (where Y is: A, {alpha}; M, µ; P, {pi}) GST of subunit type 1 from x (where x is: h, human; r, rat). Back

4 L. Jia; K. Tew and V. Findlay; J. Liu and M. Waalkes, personal communications. Back

Received 11/25/02; revised 1/30/03; accepted 2/12/03.


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 Results
 Discussion
 References
 

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