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Vol. 1, 11-20, November 2001     Molecular Cancer Therapeutics
© 2001 American Association for Cancer Research

Dual Role of Glutathione in Modulating Camptothecin Activity: Depletion Potentiates Activity, but Conjugation Enhances the Stability of the Topoisomerase I-DNA Cleavage Complex1

Michael P. Gamcsik2, Mohit S. Kasibhatla, David J. Adams, James L. Flowers, O. Michael Colvin, Govindarajan Manikumar, Mansukh Wani, Monroe E. Wall, Glenda Kohlhagen and Yves Pommier

Department of Medicine, Duke Comprehensive Cancer Center, Duke University Medical Center, Durham, North Carolina 27710 [M. P. G., M. S. K., D. J. A., J. L. F., O. M. C.]; Research Triangle Institute, Research Triangle Park, North Carolina 27709 [G. M., M. W., M. E. W.] and Laboratory of Molecular Pharmacology, National Cancer Institute, NIH, Bethesda, Maryland 20892-4255 [G. K., Y. P.]


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Depletion of glutathione (GSH) in MCF-7 and MDA-MB-231 cell lines by pretreatment with the GSH synthesis inhibitor buthionine sulfoximine potentiated the activity of 10,11-methylenedioxy-20(S)-camptothecin, SN-38 [7-ethyl-10-hydroxy-20(S)-camptothecin], topotecan, and 7-chloromethyl-10,11-methylenedioxy-20(S)-camptothecin (CMMDC). The greatest potentiation was observed with the alkylating camptothecin CMMDC. Buthionine sulfoximine pretreatment also increased the number of camptothecin-induced DNA-protein cross-links, indicating that GSH affects the mechanism of action of camptothecin. We also report that GSH interacts with CMMDC to form a stable conjugate, 7-(glutathionylmethyl)-10,11-methylenedioxy-20(S)-camptothecin (GSMMDC), which is formed spontaneously in buffered solutions and in MCF-7 cells treated with CMMDC. GSMMDC was synthesized and found to be nearly as active as 10,11-methylenedioxy-20(S)-camptothecin in a topoisomerase (topo) I-mediated DNA nicking assay. The resulting topo I cleavage complexes were remarkably stable. In cell culture, GSMMDC displayed potent growth-inhibitory activity against U937 and P388 leukemia cell lines. GSMMDC was not active against a topo I-deficient P388 cell line, indicating that topo I is its cellular target. Peptide-truncated analogues of GSMMDC were prepared and evaluated. All three derivatives [7-({gamma}-glutamylcysteinylmethyl)-10,11-methylenedioxy-20(S)-camptothecin, 7-(cysteinylglycylmethyl)-10,11-methylenedioxy-20(S)-camptothecin, and 7-(cysteinylmethyl)-10,11-methylenedioxy-20(S)-camptothecin] displayed topo I and cell growth-inhibitory activity. These results suggest that 7-peptidyl derivatives represent a new class of camptothecin analogues.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The camptothecins (Fig. 1) are a class of chemotherapeutic agents that act by inhibiting topo3 I (1, 2). topo I mediates relaxation of DNA in a process essential to replication and transcription. The relaxation mechanism involves cleavage of one-strand of DNA resulting in the formation of a covalent DNA-enzyme intermediate. Camptothecins stabilize this covalent intermediate by preventing topo I-mediated religation of the DNA strand (1). Whereas camptothecins are active against a wide range of solid tumors, resistance can be a significant hindrance to therapy. Reported mechanisms of camptothecin resistance include down-regulation or mutation of topo I, reduced drug uptake, and defects in apoptotic response (3). Recent work from this laboratory with cultured MCF-7 and MDA-MB-231 cells showed that a decrease in the pH of the extracellular medium potentiated camptothecin activity. This enhancement correlated with a decrease in intracellular GSH and was greatest for CMMDC (4). CMMDC is an interesting hybrid that combines the ability to bind and stabilize the topo I-DNA complexes typical of the camptothecins with a DNA-alkylating capacity characteristic of the nitrogen mustards or nitrosoureas (5, 6). GSH plays an important role in resistance to alkylating agents by a number of processes including inactivation by formation of drug-GSH conjugates in spontaneous or enzyme-catalyzed reactions (7). The pH-induced GSH depletion, therefore, may account for potentiation of CMMDC if a conjugate is readily formed. Other investigators have reported that GSH may also play a role in sensitivity to other nonalkylating camptothecins (8-10). Those studies did not determine whether GSH interacts with these agents or topo I.



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  		Fig. 1. The structure of the lactone and carboxylate forms of camptothecin and analogues.

 
This investigation sought to further define the role of GSH in camptothecin sensitivity. We found that GSH depletion increases the growth-inhibitory activity and the amount of DPCs formed by a number of camptothecin analogues. GSH appears to interact directly only with CMMDC to form the stable conjugate GSMMDC. Interestingly, we found that this conjugate retains growth-inhibitory activity and that its cleavage complexes are remarkably resistant to salt-induced reversal.

We also report the structure-activity relationship of camptothecin-peptide conjugates CGMMDC, ECMMDC, and CysMMDC and propose that 7-peptidyl camptothecins may be an important new class of camptothecin analogues.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
GSH was obtained from Sigma Chemical Co. (St. Louis, MO). Monobromobimane was obtained from Cal Biochem (La Jolla, CA), and the C18 solid-phase extraction cartridges are a product of Burdick and Jackson (Muskegon, MI) and were obtained from VWR Scientific (Suwanee, GA).

Human topo I was expressed in Sf9 insect cells using a recombinant baculovirus and purified as described previously (11).

Organic Syntheses
CMMDC and MDC were synthesized by previously published methods (12, 13).

7-Hydroxymethyl-10,11-methylenedioxy-20(S)-camptothecin. To a mixture of MDC (1.0 g; 2.55 mmol), methanol (30 ml), and H2O (25 ml), 75% H2SO4 (25 ml) was added dropwise, and then FeSO4·7H2O (0.8 g; 2.9 mmol) was added. To the ice-cold mixture, 30% H2O2 (5 ml; 2.2 mmol) was added dropwise. The mixture was stirred at room temperature for 16 h and poured onto ice/H2O, and the precipitate was collected, washed with H2O, and dried. The crude product was recrystallized using hot dimethylformamide to give an off-white powder (830 mg; 77%): 250 MHz 1H NMR (DMSO-d6, {delta}): 0.87 (t, 3, H-18), 1.84 (m, 2, H-19), 5.10 (d, 2, 7-CH2-OH), 5.30 (s, 2, H-5), 5.41 (s, 2, H-17), 5.72 (t, 1, 7-CH2OH), 6.27 (s, 2, -O-CH2-O-), 6.47 (s, 1, 20-OH), 7.23 (s, 1, H-14), 7.49 (s, 2, H-9 and H-14), MS m/z 422 (M+).

7-Bromomethyl-10,11-methylenedioxy-20(S)-camptothecin. A mixture of 7-hydroxymethyl-10,11-methylenedioxy-20(S)-camptothecin (500 mg; 1.2 mmol), HBr (8 ml), and 1 drop of concentrated H2SO4 was heated at 80°C-90°C for 18 h. Removal of HBr under reduced pressure followed by crystallization of the product from CHCl3/methanol yielded the title compound as a light orange powder (450 mg; 78%): 250 MHz 1H NMR (DMSO-d6, {delta}): 0.86) (t, 3, H-18), 1.86 (m, 2, H-17), 5.23 (s, 4, H-5 and 7-CH2Br), 5.36 (s, 2, H-17), 6.30 (s, 2, -O-CH2-O-), 6.49 (s, 1, 20-OH), 7.22 (s, 1, H-14), 7.58 (s, 1H, H-12), 7.74 (s, 1, H-9), MS m/z 486 (M+).

GSMMDC. To a solution of 7-bromomethyl-10,11-methylenedioxy-20(S)-camptothecin (115 mg; 0.24 mmol) in slightly warm (45°C) dimethylformamide (8 ml) was added a slight excess of GSH (100 mg) in H2O (1 ml) under stirring. After 1 h, the precipitated conjugate was collected, washed five times with water, and dried to give a beige powder [148 mg; 88%; MS m/z 734 (M+ +23)]. The same conjugate was also obtained, albeit in low yield, from the corresponding 7-chloromethyl analogue.

ECMMDC. The title compound was prepared according to the procedure for GSMMDC, except that {gamma}-glutamylcysteine was used. Rapid filtration of the precipitated product yielded an off-white powder [86%; MS m/z 653 (M+ +1)].

CGMMDC. The title compound was prepared according to the procedure for GSMMDC, except that cysteinyl-glycine was used. The product was obtained as a light brown powder [82%; MS m/z 583 (M+ +1)].

CysMMDC. The title compound was prepared according to the procedure for GSMMDC, except that cysteine was used. The product was obtained as an off-white powder [91%; MS m/z 526 (M+ +1)].

Cell Lines
The parental MCF-7 breast adenocarcinoma cell line was obtained from the Karmanos Cancer Institute. The MDA-MB-231 cells were obtained from American Type Culture Collection (Manassas, VA). Both cell lines were maintained in DMEM containing 2 g/liter glucose and 10% fetal bovine serum.

The U937 line was obtained from the Duke Comprehensive Cancer Center Shared Cell Culture Facility. These cells were maintained in RPMI 1640 containing 10% fetal bovine serum.

The murine P388 murine leukemia cell line and its camptothecin-resistant, topo I-deficient subline, P388R (14), were a gift from M. R. Mattern and R. K. Johnson (Glaxo Smith-Kline, King of Prussia, PA).

Growth Inhibition Assays
A propidium iodide-based assay was used to assess growth inhibition in the MCF-7 and MDA-MB-231 cell lines by a procedure published previously (15). Cells were exposed to agents for 2 cell doublings and switched to drug-free media for 1 doubling before the addition of propidium iodide.

For the U937 cell line, cells were exposed to agents for 72 h, and then a formazan-based assay using Owen’s and phenazine methosulfate reagents (Promega, Madison, WI) was used to assess growth inhibition.

For both assays, absorbance was determined in 96-well plates using a BioTek EL340 plate reader. Dose-response curves were constructed, and IC50 values were determined using Table Curve 2D software (SPSS).

Effect of BSO on Toxicity and Formation of the Cleavage Complex
GSH was assayed in all cell lines by HPLC analysis of monobromobimane-treated cells (16). For depletion studies, cells were exposed to medium containing 50 µM BSO for 24 h. The activity of the camptothecins was evaluated using calcein-AM growth inhibition assay (15). Cells were exposed to drug for 2 cell doublings and then exposed to drug-free medium for 1 additional cell doubling (15). The IC50 was determined in control cells and in cells pretreated for 24 h with 50 µM BSO.

The DNA-protein cross-linking assay was performed according to previously published procedures (17, 18). Briefly, after drug treatment and scraping in ice-cold HBSS, cells were irradiated with 30 Gy. DPCs were analyzed under non-deproteinizing and DNA-denaturing conditions using protein-absorbing filters (polyvinylchloride-acryl copolymer filters; 0.8 µm pore size; Gelman Science, Ann Harbor, MI) and SDS lysis solution (17). The DNA was eluted from filters with tetrapropylammonium hydroxide-EDTA (pH 12.1) without SDS at a flow rate of 0.02–0.04 ml/min. DPCs were calculated according to the bound to one terminus model formula (18):

where r is the retention for drug-treated cells, and r0 is the retention for the untreated cells.

NMR Analysis of Ring-opening Kinetics
NMR spectroscopy was performed on a Varian Unity 500 MHz NMR spectrometer. Each spectrum was acquired in 2.25 min periodically during 1 h. Topotecan (1 mM) was dissolved in 0.05 M sodium phosphate in deuterated water, and the rate of lactone ring opening was determined by following the intensity of the separate methyl proton resonances in the ring-closed (0.95 ppm) and ring-opened (1.06 ppm) forms. Reactions were carried out in the absence and presence of 10 mM GSH. In one experiment, 1.5 mg of GSH S-transferase (porcine liver; Sigma Chemical Co.) was added to the 0.7-ml sample. Ring-opening rates were obtained by fitting the lactone intensity versus time, assuming a reversible first-order reaction using SigmaPlot (Jandel Scientific, Corte Madera, CA). The reported rate constant is for the forward ring-opening rate.

HPLC Analysis of the Conjugation
HPLC analyses were performed on a Waters HPLC system using a 4.6 x 250-mm Zorbax RX C18 column (Hewlett-Packard). The mobile phases were acetonitrile and 2% triethylamine adjusted to pH 5.5 with glacial acetic acid (19). Initially, the column was eluted with 25% acetonitrile and 75% triethylamine. This was changed linearly over the first 6 min to 40% acetonitrile. The next minute brought the eluant to 100% acetonitrile, which was maintained for 4 min. Finally, the gradient was returned to the initial conditions. The eluent was analyzed by UV spectroscopy using a Waters PDA 996 photodiode array detector scanning between 300 and 500 nm and by spectrofluorometry using a Waters 474 monitor with an excitation wavelength of 382 nm and an emission wavelength of 421 nm. Fig. 3 shows the chromatogram obtained by monitoring the eluant at 382 nm.



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  		Fig. 3. HPLC chromatograms of (a) 20 µM CMMDC after incubation for 20 min in 0.05 M phosphate (pH 7.4) at 37°C; (b) the sample described in a 4 min after the addition of 5 mM GSH; and (c) acidification of the sample described in b and incubation at 37°C for 30 min. Peaks are labeled as follows: A, CMMDC-lactone; B, CMMDC-carboxylate; C, GSMMDC-lactone; and D, GSMMDC-carboxylate.

 
For initial HPLC analysis, a solution of CMMDC in 0.05 M phosphate buffer was prepared by diluting a 500 µM stock solution in DMSO to achieve a drug concentration of 20 µM. Using a higher stock solution concentration of CMMDC in DMSO resulted in drug aggregation in the buffer and much slower ring opening. This solution was analyzed by HPLC using the conditions noted above. To this, a GSH solution was added in the phosphate buffer to a final concentration of 5 mM. This solution was analyzed by HPLC. The pH of this reaction mixture was lowered to pH 4.5 using 2 M acetic acid, and the solution was incubated for 30 min at 37°C and analyzed again by HPLC.

Preparation of Samples for Mass Spectrometry
The GSH and CMMDC reaction was repeated, and additional steps were performed to purify the sample for mass spectrometric analysis. A solution of 50 µM CMMDC and 10 mM GSH, pH 7.4, was incubated at 37°C for 3 h. After 3 h, the reaction mixture was passed through a 0.4 µm filter to remove any undissolved drug. Further purification was performed using a C18 solid-phase extraction cartridge. Solid-phase cartridges were prepared by washing with acetonitrile, followed by extensive washing with 0.2% acetic acid. The reaction mixture was loaded onto the cartridge, washed with 0.2% acetic acid, and eluted using a 30% acetonitrile-water solution. The eluant was dried, redissolved, and analyzed by both HPLC and mass spectrometry. The sample for matrix-assisted laser desorption ionization mass spectrometry was diluted in acetonitrile:water (1:1) containing 0.5% trifluoroacetic acid. The matrix used for analysis was {alpha}-cyano-4-hydroxycinnamic acid. Matrix-assisted laser desorption ionization mass spectrometry was performed on a PerSeptive Biosystems Voyager DE spectrometer.

Kinetics of Conjugate Formation
A 5 mM GSH solution in PBS was prepared, and the pH was adjusted to pH 7.4 with NaOH. To this was added a 10 mM solution of CMMDC in DMSO to achieve a final concentration of 20 µM. The solution was filtered through a 0.4 µm filter before incubation at 37°C. Periodically, 200-µl aliquots were removed, the reaction was stopped by the addition of 0.1 M HCl, and the samples were rapidly frozen and stored at -135°C. Thawed samples were analyzed by HPLC. With GSH in excess, the data were analyzed assuming irreversible pseudo first-order kinetics.

Cell Studies
MCF-7 cells were cultured with and without 1 µM MDC or CMMDC at 37°C for 1 h. Afterward, drug-treated and drug-free media were removed, frozen, and lyophilized. The cells in the flask were rapidly washed with ice-cold Dulbecco’s PBS and then immediately treated with 0.5 M perchloric acid. The cell debris was removed from the flask surface with a cell scraper. The precipitated protein was removed by centrifugation. The pH of the supernatant was adjusted to between pH 3 and pH 4 with 5 M KOH. The potassium perchlorate precipitate was removed by centrifugation, and the supernatant was applied to a solid-phase extraction cartridge and treated as described above for the preparation of the mass spectrometry samples. The eluant from the 30% acetonitrile wash was used for HPLC analysis and purification of the conjugate.

The lyophilized media were dissolved in a minimal volume of 0.5 M perchloric acid, and the extraction procedure was repeated for the media.

DNA Nicking Assay
Assays were performed as described previously (20-22). In Figs. 5 and 6, a pBluescript DNA fragment was used, and in Figs. 4 and 8, an oligonucleotide containing a single topo I cleavage site was used. For both DNA fragments, labeling was at the 3'-end for unambiguous determination of the topo I-mediated DNA cleavage sites. Reactions with human topo I in the presence and absence of drug were performed at 25°C. Reactions were stopped by the addition of 0.5% SDS.



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  		Fig. 5. Image of a polyacrylamide gel showing the effects of MDC, GSMMDC, and CPT on topo I-mediated DNA cleavage. Reactions were performed for 30 min at 25°C and stopped by the addition of 0.5% SDS. The concentrations of added analogues in each triangle are 0.01, 0.1, 0.3, 1, 3, and 10 µM. Numbers to the right correspond to position of cleavage sites (22).

 


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  		Fig. 6. Stability of the topo I cleavage complexes induced by 1 µM MDC, GSMMDC, and CPT. A, image of the polyacrylamide gel described in Fig. 5, except that the reaction mixtures were incubated for 15 min at 25°C, and then an aliquot was removed (time 0), and 0.35 M NaCl was added. The time after addition of NaCl is given above each lane. Reactions were stopped by adding 0.5% SDS. DNA fragments are indicated by the arrows. Symbols ({blacksquare} and {circ}) refer to B. B, quantitation of the data shown in A for each of the three camptothecin derivatives. Squares and circles correspond to cleavage sites 92 and 117 in Fig. 5 and in A.

 


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  		Fig. 4. Mass spectrum of GSMMDC isolated from a cell extract.

 


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  		Fig. 8. Stability of topo I cleavage complexes induced by 1 µM CPT, GSMMDC, ECMMDC, CGMMDC, and CysMMDC. Reactions were incubated for 15 min at 25°C, and then an aliquot was removed and heated to 65°C for the length of time indicated above each lane. Asterisks indicate weak topo I cleavage sites observed with the camptothecin-peptidyl conjugates.

 
Stability of the cleavage complexes was assessed using either salt- or heat-induced reversal. Cleavage complexes were formed by incubating topo I, drug, and DNA (or oligonucleotide) for 15 min at 25°C. For salt-induced reversal, 0.35 M NaCl was added for the indicated times at 25°C to enable religation, and then reactions were stopped with 0.5% SDS. For heat-induced reversal, the reaction mixtures were heated to 65°C for the indicated times, and the reactions were stopped with 0.5% SDS. Loading buffer was then added to the samples, which were loaded onto 20% polyacrylamide gels containing 7 M urea. Imaging and quantitation were performed using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of BSO on the Toxicity of the Camptothecins. To assess the contribution of GSH in the sensitivity to the camptothecins, cells were pretreated with BSO before the cytotoxicity assays. BSO is an inhibitor of {gamma}-glutamylcysteine synthetase, the rate-limiting enzyme in GSH biosynthesis (23). Treatment of MCF-7 and MDA-MB-231 cells for 24 h with 50 µM BSO led to a reduction in GSH levels of at least 75% in the cell lines tested (Table 1) without affecting cell growth. Pretreatment with BSO also resulted in lower IC50 values for each of the analogues tested in all of the cell lines (Table 1). GSH depletion had the greatest effect on CMMDC, the alkylating camptothecin analogue.


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  		Table 1 Effect of BSO on GSH levels and growth-inhibitory activity

 
Effect of BSO on the Formation of topo I-DNA Complexes. To determine whether GSH depletion directly affected the stabilization of the camptothecin-topo I cleavage complex, the levels of camptothecin-induced DPCs were measured in control cells and in cells pretreated with BSO. These DPCs correspond to topo I cleavage complexes (17). Pretreatment of MDA-MB-231 cells with BSO increased the number of DPCs induced by camptothecin analogues CMMDC and MDC (Fig. 2).



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  		Fig. 2. DPCs produced by CMMDC (triangles) and MDC (squares) without (closed symbols) or with (open symbols) BSO pretreatment. DPCs were measured in MDA-MB-231 cells treated for 1 h with CMMDC or MDC. BSO pretreatments were 24 h long, with the camptothecins added during the last hour. The data show the result of a single experiment. Similar results were obtained in independent experiments.

 
Effect of GSH on Camptothecin Chemistry. Using both NMR spectroscopy and HPLC methods, we found that GSH does not affect the rate of lactone ring opening for topotecan or MDC (data not shown). Similarly, no covalent conjugate was detected between these camptothecin derivatives and GSH in the absence or presence of GSH S-transferase.

By contrast, the alkylating camptothecin analogue CMMDC formed a covalent conjugate with GSH. Fig. 3a shows the HPLC chromatogram of a solution of 20 µM CMMDC approximately 20 min after dissolution in phosphate buffer, pH 7.4, 37°C. Two peaks, A and B, with retention times of 9.90 and 6.92 min were observed, corresponding to the lactone and carboxylate forms of CMMDC, respectively. After mixing this solution with GSH, two new peaks, C and D, appeared with retention times of 3.29 and 3.04 min (Fig. 3b). Both peaks C and D had UV spectra typical of camptothecin, with local absorption maxima near 370 and 390 nm. These data suggest that the addition of GSH to CMMDC results in the conversion of CMMDC to two new species, represented by peaks C and D. After acidification of the reaction mixtures and incubation for 30 min at 37°C, analysis by HPLC showed significant conversion from peak D to peak C (Fig. 3c), suggesting that the lactone was in equilibrium with its ring-opened form.

Several additional results suggest that these new species result from a reaction between GSH and CMMDC: (a) there were no additional peaks evident in the chromatogram of mixtures of MDC and GSH; (b) similarly, no additional peaks were detected in solutions of CMMDC and the oxidized form of GSH (GSSG) in which the sulfhydryl is not available for reaction; and (c) an authentic sample of 7-hydroxymethyl-10,11-methylenedioxycamptothecin, a possible hydrolysis product of CMMDC, eluted with distinct retention times of 7.17 min (lactone) and 2.88 min (carboxylate). These results are consistent with the hypothesis that peaks C and D are the lactone and carboxylate forms of the conjugate GSMMDC.

Unambiguous identification of GSMMDC required isolation of a purified sample. This was obtained by solid-phase extraction of a reaction solution of CMMDC and excess GSH by a procedure outlined in "Materials and Methods." The product of this purification step was isolated under acidic conditions, and analysis by HPLC revealed a single peak with a retention time identical to peak C. Mass spectroscopic analysis of this purified product revealed that the major molecular ion was at m/z 712, the expected molecular mass of GSMMDC (data not shown).

In all of the HPLC analyses of solutions of the parent CMMDC, the carboxylate form of this analogue was detected only when dilute (<1 mM) samples of CMMDC in DMSO were further diluted into buffer. At higher concentrations, the ring opening was slow. This may be due to the formation of self-aggregates in solution, as has been observed for MDC (24). Formation of these aggregates may stabilize the lactone form and slow the rate of ring opening.

Rate of GSH Conjugation with CMMDC. After identifying the conjugate by mass spectroscopy, the rate of reaction between GSH and CMMDC was measured by periodically analyzing the mixture by HPLC. Sufficient CMMDC to prepare a 20 µM solution was added to a solution of 5 mM GSH in PBS at pH 7.4. Because the hydrophobic CMMDC may form a microsuspension in water, the solution was filtered to remove undissolved analogue, which would complicate the kinetic analysis. The concentration of CMMDC in the resulting solution was <20 µM due to the removal of some suspended solid plus unavoidable binding of the drug to the filtration membrane. The CMMDC concentration in the reaction mixture was estimated at 7 µM by UV analysis. The reaction mixture was sampled periodically and analyzed by HPLC as described in "Materials and Methods."

The parent compound CMMDC is weakly fluorescent. On conjugation with GSH, the fluorescence increases significantly. The lower fluorescence in CMMDC may be due to quenching by the chlorine atom and/or formation of aggregates by this hydrophobic compound in aqueous solution (24). In contrast, the UV spectrum is less affected. Therefore, reaction kinetics were obtained by monitoring the HPLC eluant at 382 nm. Because GSH is present in large excess, pseudo first-order kinetics is assumed, and the best exponential fit to the data yielded a rate constant for the appearance of GSMMDC of 0.133 min-1, whereas the rate constant for the disappearance of peak A, CMMDC, was 0.165 min-1. The rate constants should be identical, but due to the rapid nature of the reaction at 37°C, these values are likely within the error of the determination. These results indicate that the formation of GSMMDC is rapid even in the absence of enzyme catalysis.

Detection of GSMMDC in Cells. To determine whether GSMMDC conjugates form in living cells, MCF-7 breast cancer cells were treated with 1 µM CMMDC for 1 h. Extracts were prepared from both the media and cells. HPLC analysis of a cell extract demonstrated the presence of GSMMDC (data not shown). Spiking these extract solutions with a sample of authentic GSMMDC enhanced only the peak thought to be the conjugate. In addition, this peak was absent in untreated and MDC-treated cells and media. Finally, this peak was collected as it eluted from the HPLC column and analyzed using mass spectrometry. The mass spectrum (Fig. 4) shows the molecular ion at 712 a.m.u., confirming the presence of GSMMDC, the camptothecin-GSH conjugate, in both cells and media treated with CMMDC.

Activity of GSMMDC. Identification of GSMMDC within cells treated with CMMDC led to an investigation of the ability of GSMMDC to inhibit topo I. This required preparation of purified samples of GSMMDC. Attempts to prepare larger quantities of GSMMDC from CMMDC in organic solvent produced low yields and, when prepared in aqueous buffer solution with an excess of GSH, were difficult to purify. Therefore, GSMMDC was synthesized in milligram quantities from 7-bromomethyl-10,11-methylenedioxycamptothecin as described in "Materials and Methods." We tested the ability of GSMMDC to inhibit topo I in DNA nicking assays (6, 21, 22). The results shown in Fig. 5 indicate that GSMMDC is less active than MDC or CPT in producing DNA cleavage in pSK DNA but has a similar distribution of the topo I cleavage sites. However, the topo I cleavage complexes formed in the presence of GSMMDC were relatively resistant to salt-induced reversal as indicated by the results in Fig. 6.

The conjugate was then tested for biological activity against breast cancer and leukemia cell lines. Table 1 shows low activity for GSMMDC against both human breast cancer cell lines tested. However, GSMMDC showed growth-inhibitory activity against the human U937 and mouse P388 leukemia cell lines (Table 2). This activity was greatly attenuated in a camptothecin-resistant P388 line known to lack topo I activity (21, 25), indicating that GSMMDC is a specific topo I poison.


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  		Table 2 IC50 for MDC and 7-peptidyl-MDC analogues (nM)

 
Activity of ECMMDC, CGMMDC, and CysMMDC. To probe the determinants of the peptide structure that influence topo I inhibition and biological activity, the dipeptides ECMMDC and CGMMDC and the amino acid conjugate CysMMDC were prepared. The topo I-inhibitory activities of the 7-peptidyl derivatives were compared to MDC, EMDC, and CMMDC using an oligonucleotide substrate containing a single high-affinity topo I cleavage site (Fig. 7). The single high-affinity site facilitates the quantitative analysis. As with the pSK substrate, GSMMDC induced strand breaks in the oligonucleotide but with a reduced activity compared with MDC. The amino acid derivative CysMMDC exhibited comparable or slightly lower activity than GSMMDC, whereas the dipeptides exhibited lower activity (Figs. 7 and 8).



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  		Fig. 7. topo I-mediated DNA cleavage in the presence of peptide-MDC conjugates. A, sequence of the 22-mer oligonucleotide, with the high-affinity topo I cleavage shown by a caret between the central T and G bases in the sequence. Labeling of the upper strand (scissile) strand with [{alpha}-32P]cordycepin is schematically represented as 32P-A. Note that the 3'-end label (cordycepin) introduces an additional A residue into the sequence. B, image of a polyacrylamide gel showing the effects of the compounds abbreviated in Fig. 1. Drug concentrations of added analogues are 0.01, 0.03, and 0.1 µM in each triangle.

 
Similar to the salt-induced reversal results (Fig. 6), the topo I cleavage complexes induced by GSMMDC also demonstrated increased resistance to heat-induced reversal compared with CPT (Fig. 8). Although CysMMDC and CGMMDC induced fewer strand breaks, the cleavage complexes formed were more resistant to heat-induced reversal than those observed with CPT (Fig. 8). The dipeptide ECMMDC showed the lowest activity and was most susceptible to reversal of all the 7-peptidyl analogues tested. However, the cleavage complexes were more stable than the ones formed in the presence of CPT, despite the greater activity of CPT. All of the peptide conjugates induced an additional cleavage site indicated by the asterisks in Fig. 8.

CysMMDC also displayed low biological activity as GSMMDC against the MCF-7 cell line (Table 2). The dipeptides were slightly more active in growth inhibition of these cells. All of the 7-peptidyl derivatives were more active against the U937 leukemia cells. In this case, the growth-inhibitory activity of GSMMDC and that of the dipeptides were similar and comparable with that of SN-38. The amino acid conjugate, CysMMDC, was about 5-fold less active (Table 2).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Earlier work from this laboratory showed that a reduction in extracellular pH potentiates the toxicity of the camptothecins (4). This may be due, in part, to the increased stability of the active lactone form of the camptothecins at lower pH (Fig. 1). In addition, the reduction in extracellular pH resulted in a decrease in the intracellular GSH concentration (4). The pH-induced potentiation was the greatest for CMMDC, the alkylating camptothecin. Because GSH has long been implicated in resistance to alkylating agents (7), the depletion of GSH may contribute to the increase in toxicity of CMMDC. However, the activities of camptothecin analogues that are not alkylators were also enhanced by pH-induced depletion of cellular GSH.

To elucidate whether the potentiation of these agents was due to GSH depletion or other pH-induced effects, we used the {gamma}-glutamylcysteine synthetase inhibitor BSO, which has a high specificity for this enzyme and depletes intracellular GSH. Potentiation of toxicity by pretreatment with BSO provides strong evidence that GSH plays a central role in sensitivity to a particular drug. For all cell lines, BSO pretreatment reduced the intracellular GSH content by at least 75% without affecting cell growth (Table 1). For camptothecin and all of the analogues tested, this depletion decreased the IC50 between 2- and 13-fold. The alkylating CMMDC displayed the greatest reduction in IC50, which is consistent with our earlier observations on the effect of acidic pH (4).

The potentiation of the activity of the camptothecins by the BSO-mediated depletion of GSH is consistent with previous reports of BSO-mediated enhancement of CPT-11 activity in a lung fibroblast cell line (10). In addition, BSO was effective in increasing CPT-11 and SN-38 activity in a cisplatin-resistant subline of human ovarian cancer but had no effect on the parental line (9). Consistent with these results is the observation of increased GSH levels in a glioma cell line selected for resistance to CPT-11 (8). In contrast to these observations, however, others did not observe an increase in toxicity of camptothecin with GSH depletion in several human leukemic cell lines (26).

Our observations presented in Table 1 indicate a role for GSH in modulating the cytotoxicity of the camptothecins in breast cancer cells. These results may be explained by an increased susceptibility of these BSO-treated cells to cytotoxins in general. For example, formation of reactive oxygen species has been reported in camptothecin-treated cells (27) and with other anticancer therapies (28). The levels of reactive oxygen species would be increased by BSO-mediated GSH depletion (28). However, the observed increases in DNA-protein cross-linking with BSO pretreatment shown in Fig. 2 suggest that GSH also affects the ability of camptothecins to act at their cellular target; i.e., topo I. The mechanism by which GSH exerts its influence is not known. Firstly, GSH does not appear to alter the rate of lactone ring opening or change the equilibrium ratio of lactone:ring-opened carboxylate. Secondly, GSH does not appear to be involved with maintaining the enzyme in a reduced form. Lastly, except for CMMDC, GSH does not appear to form conjugates with the camptothecins even in the presence of GSH S-transferase.

With CMMDC, we observed the formation of the conjugate GSMMDC, which is rapid in buffer solutions (t1/2 ~ 5 min) and likely to be just as rapid in cells. The conjugate was detected both in cell extracts and in the surrounding media, suggesting that its formation occurs under physiological conditions in the cell and that the conjugate is exported. Interestingly, the addition of a charged GSH molecule to the 7 position of the camptothecin molecule did not prevent stabilization of the DNA-topo I cleavage complexes in DNA nicking assays using two different substrates. Together, our results indicate that GSH conjugation of the camptothecin does not serve to inactivate CMMDC as is found for other alkylating agents. A possible mechanism to explain our results with both alkylating and nonalkylating camptothecins is a GSH-mediated efflux of the camptothecin analogues known to occur by a number of processes (29-34) leading to resistance (35-38).

In contrast to the possible efflux of GSMMDC from the cells is the finding that the presence of the bulky tripeptide at the 7 position of the camptothecin molecule does not prevent the binding of this analogue in the DNA-topo I complex. In fact, topo I cleavage complexes were observed in the presence of GSMMDC, and purified topo I with either the DNA or oligonucleotide substrate was remarkably stable. To determine whether the demonstrated enzyme inhibition activity results in a biological response, the growth-inhibitory activity of GSMMDC was tested against several cell lines. As shown in Table 1, the activity against both human breast cancer cell lines tested was low. This was not unexpected because the polar peptide linkage would be expected to limit transport of GSMMDC across the cell membrane. However, with the leukemia cell lines, the potency of GSMMDC was comparable with that of the other camptothecins. The lack of growth inhibition by GSMMDC against the topo I-deficient P388R cell line (Table 2) indicates that topo I is the primary cellular target of GSMMDC.

Because we found that GSMMDC was an active topo I inhibitor, three other analogues containing subsets of the amino acids in GSH were synthesized. The dipeptides CGMMDC and ECMMDC are produced by omitting the NH2- and COOH-terminal glutamyl and glycyl residues, respectively (see Fig. 1). Deletion of these single amino acids resulted in a reduction of the topo I-inhibitory activity, with the dipeptide ECMMDC showing the greatest change in activity relative to GSMMDC (Fig. 7). However, these dipeptides maintain biological activity against U937 cells (Table 2). Removal of both terminal amino acids to produce CysMMDC resulted in a modest decrease in topo I-inhibitory activity but lower biological activity. Of the amino acid and peptide conjugates, the intact GSH tripeptide appears to form the most stable topo I cleavage complexes and to exhibit significant biological activity. It has been reported previously that the potency of camptothecins was directly related to the stability of topo I cleavage complexes (39). The current data (Figs. 6 and 8) indicate that this may not be the case for the 7-peptidyl camptothecins. Furthermore, because these analogues all possess polar substituents at the 7 position, the relationship between the number and/or stability of cleavage complexes formed and the biological activity may be obscured by differential transport of these peptidyl conjugates through the cell membrane. Masking some of the polar groups of the amino acid and peptide derivatives may possibly improve the activity of all of these analogues. Future studies may also include increasing the peptide chain length and investigating the stereochemistry of binding by substituting D-amino acids. Recently, camptothecins with lipophilic groups at the 7 position have been introduced (40, 41) with the goal of increasing lactone ring stability, membrane permeability, and DNA binding. The peptidyl camptothecins developed in our laboratories illustrate that hydrophilic substituents may offer an alternative strategy to producing effective drugs.


   Acknowledgments
 
We greatly appreciate the material and advice offered by Dr. Tao Hsieh (Department of Biochemistry, Duke University, Durham, NC) and the assistance with mass spectrometry provided by Dr. George Dubay (Department of Chemistry, Duke University). The Duke University NMR Center was established with grants from the NIH, National Science Foundation, and the North Carolina Biotechnology Center.


   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 indicate this fact.

1 Supported by National Cancer Institute Grant CA68697 (to O. M. C.), Department of Defense Grant DAMD-17-99-1-9175 (to M. P. G.), and the Duke Comprehensive Cancer Center. Back

2 To whom requests for reprints should be addressed, at Department of Medicine, Box 3843, MSRB 301, Research Drive, Durham, NC 27710 Back

3 The abbreviations used are: topo, topoisomerase; BSO, buthionine sulfoximine; CGMMDC, 7-(cysteinylglycylmethyl)-10,11-methylenedioxy-20(S)-camptothecin; CPT, 20(S)-camptothecin; CMMDC, 7-chloromethyl-10,11-methylenedioxy-20(S)-camptothecin; CysMMDC, 7-(cysteinylmethyl)-10,11-methylenedioxy-20(S)-camptothecin; DPC, DNA-protein cross-link; ECMMDC, 7-({gamma}-glutamylcysteinylmethyl)-10,11-methylenedioxy-20(S)-camptothecin; GSH, glutathione; GSMMDC, 7-glutathionylmethyl-10,11-methylenedioxy-20(S)-camptothecin; MDC, 10,11-methylenedioxy-20(S)-camptothecin; MS, mass spectrometry; NMR, nuclear magnetic resonance; HPLC, high-performance liquid chromatography. Back

Received 7/12/01; revised 8/31/01; accepted 9/ 6/01.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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