BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 735-74]
Vol. 180, No. 2, 1991 October 31, 1991
INTERACTIONS OF PLATINUM COMPLEXES WITH THIOLTRANSFERASE(GLUTAREDOXIN), IN VITRO
William W. Wells, Pamela A. Rocque, Dian-Peng Xu, Yanfeng Yang and Thomas L. Deits Department of Biochemistry, Michigan State University East Lansing, MI 48824 Received September 5, 1991
Under
anaerobic
conditions,
recombinant
pig
liver
thioltransferase
(glutaredoxin)(TT,GRX) (EC 1.8.4.1) was strongly inhibited by cis and carbo-platin and somewhat less sensitive to trans-platin, in vitro. By extrapolation to total inhibition, the ratio of platinum drug/thioltransferase was approximately 1.0 for cis and carbo-platin, but > 1.0 for
trans-platin.
When thioltransferase was not reduced, inhibition by preincubation with the
platinum complexes required molar excesses of 1,300 and 675 to one for cis-platin and transplatin, respectively or 400-500#M for 50% inhibition. The inhibition of thioltransferase at high drug concentrations in the presence of oxygen was associated with cross-linking of monomers into dimers within 5 min and, in the case of cis-platin treatment, to trimers in 20 min incubation. © 1991
Academic
Press,
Inc.
Cis-platin, cis-diamminedichloroplatinum, is one of the most successful drugs in the treatment of human malignancies especially testicular, ovarian, bladder and head and neck cancers (1,2). There is considerable evidence that DNA is the principal intracellular target of
cis-plafin (2).
However, cis-platin's effectiveness is attenuated by its toxic side effects on
various tissues, especially the kidney, suggesting a further mechanism of toxicity analogous with that of other heavy metals. It has been reported that cis-plafin reacts with sulfur-containing nucleophiles such as glutathione (3-6). However, Leyland-Jones et al. (7) demonstrated that cisplatin nephrotoxicity is not mediated by depletion of intracellular glutathione nor by inhibition of 3,-glutamyl transpeptidase.
In other studies no change was observed in kidney or liver
glutathione levels, glutathione disulfide reductase or glutathione peroxidase activity (8).
In
contrast, cis-platin-induced nephrotoxicity in rats was inhibited by buthionine sulfoximine, a
735
0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
glutathione synthesis inhibitor (9). Thus, the possible interaction of platinum complexes with glutathione or its related enzymes, in vivo, remains in doubt. Thioltransferase, also known as mammalian glutaredoxin (TF,GRX)(10,11), is a small molecular weight protein which catalyzes thiol-disulfide exchange in cells between small and large molecular weight substrates (12-15).
Until recently, specific cellular functions for
mammalian TT,GRX were confined to a putative participation in the electron transport pathway of the ribonucleotide reductase complex in calf thymus (16), but not in the rabbit bone marrow reductase system (17). In vitro studies suggest that TT,GRX is required for the GSH-dependent deiodination of T 4 to T 3 (18). In a recent communication from this laboratory (19), we reported that mammalian TT,GRX had intrinsic dehydroascorbate reductase (EC 1.8.5.1) activity, in
vitro, and we speculated that a vital cellular function of TT,GRX is to catalyze the GSHdependent regeneration of ascorbate from dehydroascorbate formed in response to oxidative stress. Therefore, a drug that significantly inhibits TT,GRX might be expected to interfere with the cellular defense against prooxidative factors in cells. Furthermore, an explanation for the effectiveness of such a drug in cancer chemotherapy may follow from the relative susceptibility of transformed cells versus normal cells to drugs whose mechanism of action include production of cytotoxic levels of oxygen free radicals. In this communication we demonstrate that under anaerobic conditions, cis-platin and carbo-platin and, to a lesser degree, trans-platin are potent inhibitors of TT,GRX, in vitro. Materials and ~thods
Glutathione disulfide reductase, isolated from yeast, glutathione, dithiothreitol, NADPH, HEPES, Tris, cis-platin, trans-plafin and carbo-platin were products of Sigma Chemical Company. Acrylamide, N,N-methylene bisacrylamide, ammonium persulfate, and SDS were from Bio-Rad. S-Sulfocysteine was prepared as described previously (10). Homogeneous recombinant pig liver TT,GRX was isolated as described previously (20). Protein was determined by the bicinchoninic acid protein assay protocol according to the manufacturer's direction (Pierce Chemical Co.) with bovine serum albumin as standard and thioltransferase amounts were determined based on the specific activity of recombinant pig liver enzyme (82 units /mg). Enzyme catalyzed reactions were analyzed using a Gilford Response II spectrophotometer as described previously (21). Glutathione disulfide reductase was assayed by the method of Massey and Williams (22). The reaction mixture contained 0.2 mM NADPH, 0.33 mM GSSG, 60 mM sodium phosphate, pH 7.5, 1 mM EDTA, and 1 mg bovine serum albumin in a volume of 0.5 ml. Interaction of Platinum Complexes With Enzymes, in vitro. Stock solutions of 0.67/zM and 6.67#M cis-platin and trans-platin and 0.54-5.4#M carbo-plafln were prepared in tripledistilled water, air was removed by repeated evacuation and the solutions were sparged with argon. Thioltransferase stored in 50mM sodium phosphate buffer, pH 7.5, 25% glycerol was incubated with 100 mM DTT in 200/~1 of sodium phosphate buffer, pH 7.5. The sample was sparged with argon and passed through a Sephadex G-25 column previously equilibrated with argon-sparged buffer. The reduced anaerobic enzyme was transferred to a Coy glovebox maintained under argon, reacted with the platinum drugs in a total volume of 50/A, removed and assayed as described previously (21) within 30-60 min. 736
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Cross-linking of Tr.GRX bv Cis-olatin and Trans-olatin. Since platinum complexes are known to cross-link guanine bases within and across DNA chains (2), we investigated whether cis-platin or trans-platin could cross-link Tr,GRX under aerobic conditions as judged by SDS PAGE analysis. TT,GRX, previously dissolved in 50% glycerol, 100 mM sodium phosphate, pH 7.5 and 1.7/~M DTT was diluted 30 fold by solutions of either 0.5 mM cis-platin or transplatin previously dissolved in 150 mM NaC1 in a volume of 36/zl. The mixture was rapidly divided into 3 aliquots of 10/~1 placed in SDS PAGE sample tubes. The tubes were incubated at 30°C for 5, 20, or 60 min. At each period, 10 #1 of twice concentrated sample buffer according to Laemmli (23) were added, placed in a boiling water bath for 3 min and loaded on a mini gel as described previously (10). The gel was fixed, and stained with silver (24).
Results and Discussion
Inhibition of Enzymes by Platinum Complexes. In the standard q'T,GRX assay (21), glutathione reductase (GR) is utilized as a coupling enzyme to monitor the progress of the dithiol-disulfide exchange reaction spectrophotometrically at 340 nm. Therefore, it was first necessary to evaluate the effect of the platinum complexes on glutathione reductase activity. In each case under aerobic conditions, up to 1 mM concentrations of the platinum complexes had no effect on glutathione reductase activity . Since GSH is known to interact with platinum complexes (3-6), incubation of the enzymes with inhibitors preceded addition of the cosubstrate, GSH. The aerobic inhibition of Tr,GRX by trans-platin and cis-platin gave irreversible fight binding plots (data not shown). The amount of TT,GRX totally inactivated by 200#M and 400
ixM trans-platin and cis-platin by extrapolation was calculated to be 675 and 1,322 to 1, respectively. Carbo-platin (400/xM) caused no inhibition of pig liver TT,GRX under aerobic conditions. To investigate possible reversibility, TT,GRX, preincubated aerobically with 0.5 mM trans-platin, was run through a Sephadex G-25 gel filtration column, and the protein peak was assayed for activity. The percent inhibition before and after chromatography was equai (data not shown). Using appropriate levels of anaerobically reduced thioltransferase (7.5-30 pmoles), the experimental points representing reactions with cis-platin and carbo-platin at low platinum drug to thioltransferase ratios lay roughly on a line that extrapolated to 1.0 (Fig. 1A and B). At higher Pt/TT,GRX ratios, there was an unexplainable deviation from 1.0 stoichiometry in which a fraction of the enzyme appeared less sensitive to inhibition. We speculate that this fraction represents a highly stable conformation of Tr,GRX that either resists reduction by 100 mM DTT, or is reduced but resistant to interaction with the platinum drugs by steric protection. The experimental points for trans-platin inhibition were largely above the theoretical line for a 1:1 stoichiometry (Fig. 1C). 737
Vol. 180, No. 2, 1 991
100
80 -
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
B 100
...... =,~.
~'-",!
1
8O
l .>
I
60
60 ",,,
E(D
40
i,°
20
20
011
012
o.a
0.4 o15 o16 mol Cis-platin / mol l-r
dlI
b
o'.7 o16 o19
0'.1
012 0'.3
0'.4 0'.5
0'.6 o'.r
0.8
019
mol Carbo-platin / mol n
C 100-
80-
•
• ••
•
60E 40-
20
o
o
011
012
013
014
015
016
0.7
018
019
mol Trans-platin / mol TT
Fiaure 1. The stoichiometry of the inhibition of pig liver thioltransferase by A, cis-platin; B carbo-platin and C, trans-platin. Homogeneous thioltransferase (7.5-30 pmoles) was reduced as described in the text, reacted with the designated ratios of platinum drugs to enzyme and assayed for thiol-disulfide activity as described previously (21). Thioltransferase activity is expressed as percent of uninhibited controls for 5-12 enzyme preparations plotted against the [platinum drug]/[thioltransferase]. The points are individual experimental values and are compared with a line depicting the theoretical stoichiometry of 1.0 for total inactivation.
Cross-linking of TT,GRX by cis-platin and trans-Dlatin. The time course of treatment of pig liver TT,GRX in sodium phosphate buffer, pH 7.5, with 220 #M cis-platin or
trans-pladn showed stable cross-linking leading to dimers after 5 • i n incubation at 30°C as determined by SDS PAGE (Fig. 2). Trimers were evident within 20 min only in the case of cisplatin reactivity. Although much TT,GRX remained in the monomeric form up to 60 • i n , we did not determine whether these monomers were partially or totally inhibited by a single platinum ligand, i.e., displacing a single chlorine or water ligand by a protein sulfur ligand.
Cis-platin and carbo-platin, and to a lesser extent trans-platin, were strong inhibitors of reduced homogeneous pig liver Tr,GRX. This inhibition by preincubation is irreversible, since saturation levels of GSH in the assay mixture failed to reverse the inhibition or the accompanying cross-linking of TT,GRX subunits (data not shown). These findings, together 738
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MW
(K)
1 2 3 4 5 6 7 8
97.4 66.2 42.6 31.0
21.5 14.4
Figure 2. Pig liver TI',GRX was incubated aerobically in 3.3 mM sodium phosphate buffer, pH 7.5, 1.5% glycerol and 220 #M cis-platin for periods of 5(lane 3), 20(lane 4), and 60 min (lane 5) at 30°C according to the Materials and Methods section. Similarly, TT,GRX was incubated with 220 #M trans-platin for 5 (lane 6), 20 (lane 7) and 60 min (lane 8). Lane 1 and 2 were low molecular weight standards (Biorad) and untreated Tr,GRX, respectively. Samples were treated with 2x sample buffer (23) and separated on a 15% SDS-PAGE gel, fixed and silver strained as described previously (24). with the confirmation of the work of others (8,25) that glutathione disulfide reductase was not affected by the platinum complexes, suggest that cellular TT,GRX activity may be strongly impaired in patients during cancer chemotherapy. Other enzymes that have been suggested as participating in the clinical side effects arising from in vitro inhibition studies include malate dehydrogenase (26), liver and yeast alcohol dehydrogenase (27), glyceraldehyde 3-phosphate dehydrogenase, aldolase, and glucose-6-phosphate dehydrogenase (28), thymidylate synthetase (29), renal Na+/K+-activated and Mg2+-activated ATPases (30), glutathione peroxidase (25)
and E. coli ribonucleotide reductase (31). In the latter study cis-platin was selectively more inhibitory than trans-platin. In the present study, no significant differences were observed for the inhibitory action of cis-and carbo-platin, whereas TT,GRX was less sensitive to trans-platin. In a study of the inhibition of DNA synthesis by cis- and trans-platin in leukemia cells, YAC-1 and RADA1 (32), the discrepant relationship between the relative inhibition of DNA synthesis by the cis-and trans-platin, in vitro, and their antitumor-activity in clinical oncology was noted. The degree of inhibition of DNA synthesis by trans-platin was about the same as that by cis-platin, in vitro, in the absence of serum, but the former caused much inhibition than the latter in the presence of serum, in vitro, or in vivo.
lower
Atomic absorption
studies showed that the amount of trans-platin trapped by the serum, in vitro, is much larger than that of cis-platin. By extrapolation, it was speculated that the amount of trans-platin bound to DNA, in vivo, must be considerably smaller than that of cis-platin, thus providing an attractive explanation for the results of the antitumor potency of the two geometric isomeric forms of the drug. By analogy, TT,GRX, in vivo, is likely to be more inhibited by treatment with cis-platin as compared with trans-platin. It is well documented that the platinum complexes interact not only with DNA (2), but also with sulfur containing compounds such as GSH (3-6,33), hence it is not surprising that 739
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TT,GRX, which contains an especially strong nucleophilic thiol at cysteine-22 (34), would be inhibited irreversibly by the platinum complexes.
We found that cis-platin and trans-plafin
caused significant cross-linking of pig liver "I~,GRX as assessed by SDS PAGE and estimation of the subunit molecular weights compared with standards.
The cross-linked oligomers of
TT,GRX with the cis-platin and trans-platin were shown to be totally and irreversibly inactivated by the interaction, in vitro. It remains to be determined whether this same process occurs, in
vivo.
Others have observed cross-linking of selected proteins by the platinum complexes. For
example, reaction with a2-macroglobulin resulted in subunit cross-linking and loss of proteinase binding activity (35). In future studies, it will be valuable to assess the relative TT,GRX levels in platinum drug resistant and susceptible cell lines (36). Acknowledaments We thank Randy Kuntzman for assistance in early phases of this study not reported herein and Carol McCutcheon for typing the manuscript. This work was supported by U.S. Public Health Service Grant CA-51972 and USDA Competitive Research Grant Award 89-37120-4757. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
16. 17. 18.
Prestayko, A. W., Crooke, S. T. and Carter, S. K (eds.)(1980) Cisplatin: current status and new developments. Academic Press, New York. Pinto, A. L. and Lippard, S. (1985) Biochim. Biophys. Acta 780, 167-180. Levi, J., Jacobs, C., Kalman, S. M., McTigue, M. and Weiner, M. W. (1980) J. Pharmacol. Exp. Ther. 213,545-550. Long, D. F,, and Repta, A. J. (1981) Biopharm. Drug Dispos. 2, 1-16. Odenheimer, B. and Wolf, W.(1982) Inorganica Chimica Acta, 66, L41-L43. Berners-Priee, S. J. and Kuchel, P. W.(1990) J. Inorgan. Biochem., 38, 305-326. Leyland-Jones, B, Morrow, C., Tate, S., Urmacher, C., Gordon, C., and Young, C. W. (1983) Cancer Res. 43, 6072-6076. Maines, M. D. (1986) Biochem. J. 237, 713-721. Mayer, R. D., Lee, K-E., and Cockett, T.K. (1987) Cancer Chemother. Pharmacol., 20, 207-210. Gan, Z.-R., and Wells, W. W. (1988) J. Biol. Chem. 263, 9050-9054. Papayannopoulos, I.A., Gan, Z.-R., Wells, W. W. and Biemann, K. (1989) Biochem. Biophys. Res. Commun. 159, 1448-1454. Askelof, P., Axelsson, K., Eriksson, S., and Mannervik, B. (1974) FEBS LETTERS, 38, 263-267. Mannervik, B. and Axelsson K. (1980) Biochem. J. 190: 125-130. Ziegler, D. M. (1985) Ann. Rev. Biochem. 54, 305-329. Mannervik, B., Carlberg, I., and Larson, K. (1989) In: Dolphin, D., Poulson, R. and Avramovic, O., (eds.), Coenzymes and Cofactors Vol. III, Glutathione, Chemical, Biochemical and Medical Aspects, part A pp. 475-516. John Wiley and Sons, New York. Luthman, M., Ericksson, S, Holmgren, A., and Thelander, L.(1979) Proc. Natl. Acad. Sci. USA, 76, 2158-2162. Hopper, S., and Iurlano, D. (1989) J. Biol. Chem., 258, 13453-13457. Goswami, A., and Rosenberg, I. N. (1985) J. Biol. Chem. 260, 6012-6019. 740
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19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30 31. 32. 33. 34. 35. 36.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Wells, W. W., Xu, D-P., Yang, Y., and Rocque, P. A. (1990) J. Biol. Chem., 265, 15361-15364. Yang, Y. and Wells, W. W. (1990) J. Biol. Chem., 265,589-593. Gan Z.-R. and Wells, W. W. (1987) Anal. Biochem., 162, 265-273. Massey, V. and Williams, C.H.,Jr. (1965) J. Biol. Chem., 240, 4470-4480. Laemmli, U. K. (1970) Nature(London), 227, 680-685. Giulian, G., Moss, R. L., and Greaser, M. (1983) Anal. Biochem., 129, 277-287. Milano, G., Caldani C., Khater, R., Launay, J-M., Soummer, A-M., Nameer M., Schneider, M. (1988) Biochem. Pharmacol., 37, 981-982. Friedman, M. E., Musgrove, B., Lee, K. and Teggins, J.E.(1971) Biochim. Biophys. Acta, 250, 286-296. Friedman, M. E., and Teggins, J. E. (1974) Biochim. Biophys. Acta, 350, 263-272. Aull, J. L., Allen, R. L., Bapat, A. R., Daron, H. H., Friedman, M. E. and Wilson, J. F. (1979) Biochim. Biophys. Acta, 571,352-358. Aull, J. L. Rice, A. C. and Tebbetts, L A. (1977) Biochemistry, 16, 672-677. Daley-Yates, P. T., and McBrien, D.C.H. (1982) Chem.-Biol. Interactions, 40, 325334. Smith, S. L. and Douglas, K.T. (1989) Biochem. Biophys. Res. Commun. 162,715-723. Uchida, K., Tanaka, Y., Nishimura, T., Hashimoto, Y., Watanabe, T. and Harada, I. (1986) Biochem. Biophys. Res. Commun., 138, 631-637. Litterst, C.L., Bertolero, F., and Uozumi, J. (1986) In: McBrien, D.C.H., and Slater, T.F. (eds.), Biochemical Mechanisms of Platinum Antitumour Drugs, pp 227-254, IRL Press LTD, Oxford, England. Gan, Z-R., and Wells, W. W. (1987) J. Biol. Chem. 262, 6704-6707. Pizzo, S. V., Swaim, M. W., Roche, P. A. and Gonis, S. L.(1988) J. Inorgan. Biochem. 33, 67-76. Fichtinger-Schepman, A-M. J., Vendrik, C.P.J., vanDijk-Knijnenburg, W.C.M., deJong, W.H., van der Minnen, A.C.E., Claessen, A.M.E., van der Velde-Visser, S.D., deGroot, G., Wubs, K. L., Steerenberg, P.A., Schornagel, J. H., and Berends, F.(1989) Cancer Res., 49, 2862-2867,1989.
741
Vol. 180, No. 2, 1991 October 31, 1991
INTERACTIONS OF PLATINUM COMPLEXES WITH THIOLTRANSFERASE(GLUTAREDOXIN), IN VITRO
William W. Wells, Pamela A. Rocque, Dian-Peng Xu, Yanfeng Yang and Thomas L. Deits Department of Biochemistry, Michigan State University East Lansing, MI 48824 Received September 5, 1991
Under
anaerobic
conditions,
recombinant
pig
liver
thioltransferase
(glutaredoxin)(TT,GRX) (EC 1.8.4.1) was strongly inhibited by cis and carbo-platin and somewhat less sensitive to trans-platin, in vitro. By extrapolation to total inhibition, the ratio of platinum drug/thioltransferase was approximately 1.0 for cis and carbo-platin, but > 1.0 for
trans-platin.
When thioltransferase was not reduced, inhibition by preincubation with the
platinum complexes required molar excesses of 1,300 and 675 to one for cis-platin and transplatin, respectively or 400-500#M for 50% inhibition. The inhibition of thioltransferase at high drug concentrations in the presence of oxygen was associated with cross-linking of monomers into dimers within 5 min and, in the case of cis-platin treatment, to trimers in 20 min incubation. © 1991
Academic
Press,
Inc.
Cis-platin, cis-diamminedichloroplatinum, is one of the most successful drugs in the treatment of human malignancies especially testicular, ovarian, bladder and head and neck cancers (1,2). There is considerable evidence that DNA is the principal intracellular target of
cis-plafin (2).
However, cis-platin's effectiveness is attenuated by its toxic side effects on
various tissues, especially the kidney, suggesting a further mechanism of toxicity analogous with that of other heavy metals. It has been reported that cis-plafin reacts with sulfur-containing nucleophiles such as glutathione (3-6). However, Leyland-Jones et al. (7) demonstrated that cisplatin nephrotoxicity is not mediated by depletion of intracellular glutathione nor by inhibition of 3,-glutamyl transpeptidase.
In other studies no change was observed in kidney or liver
glutathione levels, glutathione disulfide reductase or glutathione peroxidase activity (8).
In
contrast, cis-platin-induced nephrotoxicity in rats was inhibited by buthionine sulfoximine, a
735
0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
glutathione synthesis inhibitor (9). Thus, the possible interaction of platinum complexes with glutathione or its related enzymes, in vivo, remains in doubt. Thioltransferase, also known as mammalian glutaredoxin (TF,GRX)(10,11), is a small molecular weight protein which catalyzes thiol-disulfide exchange in cells between small and large molecular weight substrates (12-15).
Until recently, specific cellular functions for
mammalian TT,GRX were confined to a putative participation in the electron transport pathway of the ribonucleotide reductase complex in calf thymus (16), but not in the rabbit bone marrow reductase system (17). In vitro studies suggest that TT,GRX is required for the GSH-dependent deiodination of T 4 to T 3 (18). In a recent communication from this laboratory (19), we reported that mammalian TT,GRX had intrinsic dehydroascorbate reductase (EC 1.8.5.1) activity, in
vitro, and we speculated that a vital cellular function of TT,GRX is to catalyze the GSHdependent regeneration of ascorbate from dehydroascorbate formed in response to oxidative stress. Therefore, a drug that significantly inhibits TT,GRX might be expected to interfere with the cellular defense against prooxidative factors in cells. Furthermore, an explanation for the effectiveness of such a drug in cancer chemotherapy may follow from the relative susceptibility of transformed cells versus normal cells to drugs whose mechanism of action include production of cytotoxic levels of oxygen free radicals. In this communication we demonstrate that under anaerobic conditions, cis-platin and carbo-platin and, to a lesser degree, trans-platin are potent inhibitors of TT,GRX, in vitro. Materials and ~thods
Glutathione disulfide reductase, isolated from yeast, glutathione, dithiothreitol, NADPH, HEPES, Tris, cis-platin, trans-plafin and carbo-platin were products of Sigma Chemical Company. Acrylamide, N,N-methylene bisacrylamide, ammonium persulfate, and SDS were from Bio-Rad. S-Sulfocysteine was prepared as described previously (10). Homogeneous recombinant pig liver TT,GRX was isolated as described previously (20). Protein was determined by the bicinchoninic acid protein assay protocol according to the manufacturer's direction (Pierce Chemical Co.) with bovine serum albumin as standard and thioltransferase amounts were determined based on the specific activity of recombinant pig liver enzyme (82 units /mg). Enzyme catalyzed reactions were analyzed using a Gilford Response II spectrophotometer as described previously (21). Glutathione disulfide reductase was assayed by the method of Massey and Williams (22). The reaction mixture contained 0.2 mM NADPH, 0.33 mM GSSG, 60 mM sodium phosphate, pH 7.5, 1 mM EDTA, and 1 mg bovine serum albumin in a volume of 0.5 ml. Interaction of Platinum Complexes With Enzymes, in vitro. Stock solutions of 0.67/zM and 6.67#M cis-platin and trans-platin and 0.54-5.4#M carbo-plafln were prepared in tripledistilled water, air was removed by repeated evacuation and the solutions were sparged with argon. Thioltransferase stored in 50mM sodium phosphate buffer, pH 7.5, 25% glycerol was incubated with 100 mM DTT in 200/~1 of sodium phosphate buffer, pH 7.5. The sample was sparged with argon and passed through a Sephadex G-25 column previously equilibrated with argon-sparged buffer. The reduced anaerobic enzyme was transferred to a Coy glovebox maintained under argon, reacted with the platinum drugs in a total volume of 50/A, removed and assayed as described previously (21) within 30-60 min. 736
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Cross-linking of Tr.GRX bv Cis-olatin and Trans-olatin. Since platinum complexes are known to cross-link guanine bases within and across DNA chains (2), we investigated whether cis-platin or trans-platin could cross-link Tr,GRX under aerobic conditions as judged by SDS PAGE analysis. TT,GRX, previously dissolved in 50% glycerol, 100 mM sodium phosphate, pH 7.5 and 1.7/~M DTT was diluted 30 fold by solutions of either 0.5 mM cis-platin or transplatin previously dissolved in 150 mM NaC1 in a volume of 36/zl. The mixture was rapidly divided into 3 aliquots of 10/~1 placed in SDS PAGE sample tubes. The tubes were incubated at 30°C for 5, 20, or 60 min. At each period, 10 #1 of twice concentrated sample buffer according to Laemmli (23) were added, placed in a boiling water bath for 3 min and loaded on a mini gel as described previously (10). The gel was fixed, and stained with silver (24).
Results and Discussion
Inhibition of Enzymes by Platinum Complexes. In the standard q'T,GRX assay (21), glutathione reductase (GR) is utilized as a coupling enzyme to monitor the progress of the dithiol-disulfide exchange reaction spectrophotometrically at 340 nm. Therefore, it was first necessary to evaluate the effect of the platinum complexes on glutathione reductase activity. In each case under aerobic conditions, up to 1 mM concentrations of the platinum complexes had no effect on glutathione reductase activity . Since GSH is known to interact with platinum complexes (3-6), incubation of the enzymes with inhibitors preceded addition of the cosubstrate, GSH. The aerobic inhibition of Tr,GRX by trans-platin and cis-platin gave irreversible fight binding plots (data not shown). The amount of TT,GRX totally inactivated by 200#M and 400
ixM trans-platin and cis-platin by extrapolation was calculated to be 675 and 1,322 to 1, respectively. Carbo-platin (400/xM) caused no inhibition of pig liver TT,GRX under aerobic conditions. To investigate possible reversibility, TT,GRX, preincubated aerobically with 0.5 mM trans-platin, was run through a Sephadex G-25 gel filtration column, and the protein peak was assayed for activity. The percent inhibition before and after chromatography was equai (data not shown). Using appropriate levels of anaerobically reduced thioltransferase (7.5-30 pmoles), the experimental points representing reactions with cis-platin and carbo-platin at low platinum drug to thioltransferase ratios lay roughly on a line that extrapolated to 1.0 (Fig. 1A and B). At higher Pt/TT,GRX ratios, there was an unexplainable deviation from 1.0 stoichiometry in which a fraction of the enzyme appeared less sensitive to inhibition. We speculate that this fraction represents a highly stable conformation of Tr,GRX that either resists reduction by 100 mM DTT, or is reduced but resistant to interaction with the platinum drugs by steric protection. The experimental points for trans-platin inhibition were largely above the theoretical line for a 1:1 stoichiometry (Fig. 1C). 737
Vol. 180, No. 2, 1 991
100
80 -
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
B 100
...... =,~.
~'-",!
1
8O
l .>
I
60
60 ",,,
E(D
40
i,°
20
20
011
012
o.a
0.4 o15 o16 mol Cis-platin / mol l-r
dlI
b
o'.7 o16 o19
0'.1
012 0'.3
0'.4 0'.5
0'.6 o'.r
0.8
019
mol Carbo-platin / mol n
C 100-
80-
•
• ••
•
60E 40-
20
o
o
011
012
013
014
015
016
0.7
018
019
mol Trans-platin / mol TT
Fiaure 1. The stoichiometry of the inhibition of pig liver thioltransferase by A, cis-platin; B carbo-platin and C, trans-platin. Homogeneous thioltransferase (7.5-30 pmoles) was reduced as described in the text, reacted with the designated ratios of platinum drugs to enzyme and assayed for thiol-disulfide activity as described previously (21). Thioltransferase activity is expressed as percent of uninhibited controls for 5-12 enzyme preparations plotted against the [platinum drug]/[thioltransferase]. The points are individual experimental values and are compared with a line depicting the theoretical stoichiometry of 1.0 for total inactivation.
Cross-linking of TT,GRX by cis-platin and trans-Dlatin. The time course of treatment of pig liver TT,GRX in sodium phosphate buffer, pH 7.5, with 220 #M cis-platin or
trans-pladn showed stable cross-linking leading to dimers after 5 • i n incubation at 30°C as determined by SDS PAGE (Fig. 2). Trimers were evident within 20 min only in the case of cisplatin reactivity. Although much TT,GRX remained in the monomeric form up to 60 • i n , we did not determine whether these monomers were partially or totally inhibited by a single platinum ligand, i.e., displacing a single chlorine or water ligand by a protein sulfur ligand.
Cis-platin and carbo-platin, and to a lesser extent trans-platin, were strong inhibitors of reduced homogeneous pig liver Tr,GRX. This inhibition by preincubation is irreversible, since saturation levels of GSH in the assay mixture failed to reverse the inhibition or the accompanying cross-linking of TT,GRX subunits (data not shown). These findings, together 738
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MW
(K)
1 2 3 4 5 6 7 8
97.4 66.2 42.6 31.0
21.5 14.4
Figure 2. Pig liver TI',GRX was incubated aerobically in 3.3 mM sodium phosphate buffer, pH 7.5, 1.5% glycerol and 220 #M cis-platin for periods of 5(lane 3), 20(lane 4), and 60 min (lane 5) at 30°C according to the Materials and Methods section. Similarly, TT,GRX was incubated with 220 #M trans-platin for 5 (lane 6), 20 (lane 7) and 60 min (lane 8). Lane 1 and 2 were low molecular weight standards (Biorad) and untreated Tr,GRX, respectively. Samples were treated with 2x sample buffer (23) and separated on a 15% SDS-PAGE gel, fixed and silver strained as described previously (24). with the confirmation of the work of others (8,25) that glutathione disulfide reductase was not affected by the platinum complexes, suggest that cellular TT,GRX activity may be strongly impaired in patients during cancer chemotherapy. Other enzymes that have been suggested as participating in the clinical side effects arising from in vitro inhibition studies include malate dehydrogenase (26), liver and yeast alcohol dehydrogenase (27), glyceraldehyde 3-phosphate dehydrogenase, aldolase, and glucose-6-phosphate dehydrogenase (28), thymidylate synthetase (29), renal Na+/K+-activated and Mg2+-activated ATPases (30), glutathione peroxidase (25)
and E. coli ribonucleotide reductase (31). In the latter study cis-platin was selectively more inhibitory than trans-platin. In the present study, no significant differences were observed for the inhibitory action of cis-and carbo-platin, whereas TT,GRX was less sensitive to trans-platin. In a study of the inhibition of DNA synthesis by cis- and trans-platin in leukemia cells, YAC-1 and RADA1 (32), the discrepant relationship between the relative inhibition of DNA synthesis by the cis-and trans-platin, in vitro, and their antitumor-activity in clinical oncology was noted. The degree of inhibition of DNA synthesis by trans-platin was about the same as that by cis-platin, in vitro, in the absence of serum, but the former caused much inhibition than the latter in the presence of serum, in vitro, or in vivo.
lower
Atomic absorption
studies showed that the amount of trans-platin trapped by the serum, in vitro, is much larger than that of cis-platin. By extrapolation, it was speculated that the amount of trans-platin bound to DNA, in vivo, must be considerably smaller than that of cis-platin, thus providing an attractive explanation for the results of the antitumor potency of the two geometric isomeric forms of the drug. By analogy, TT,GRX, in vivo, is likely to be more inhibited by treatment with cis-platin as compared with trans-platin. It is well documented that the platinum complexes interact not only with DNA (2), but also with sulfur containing compounds such as GSH (3-6,33), hence it is not surprising that 739
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TT,GRX, which contains an especially strong nucleophilic thiol at cysteine-22 (34), would be inhibited irreversibly by the platinum complexes.
We found that cis-platin and trans-plafin
caused significant cross-linking of pig liver "I~,GRX as assessed by SDS PAGE and estimation of the subunit molecular weights compared with standards.
The cross-linked oligomers of
TT,GRX with the cis-platin and trans-platin were shown to be totally and irreversibly inactivated by the interaction, in vitro. It remains to be determined whether this same process occurs, in
vivo.
Others have observed cross-linking of selected proteins by the platinum complexes. For
example, reaction with a2-macroglobulin resulted in subunit cross-linking and loss of proteinase binding activity (35). In future studies, it will be valuable to assess the relative TT,GRX levels in platinum drug resistant and susceptible cell lines (36). Acknowledaments We thank Randy Kuntzman for assistance in early phases of this study not reported herein and Carol McCutcheon for typing the manuscript. This work was supported by U.S. Public Health Service Grant CA-51972 and USDA Competitive Research Grant Award 89-37120-4757. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
16. 17. 18.
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