Using [1H,15N] heteronuclear single quantum coherance (HSQC) NMR and 15N-labeled carboplatin, 1, we show that Jurkat cells affect the rate of disappearance of the HSQC NMR peak in culture medium for this Pt2+ anticancer drug. The decay or disappearance rate constant for 1 in culture medium containing cells is k1 = kc [CO32 -] + km + ku N, where kc is the rate constant for reaction of 1 with carbonate in the medium, km is the rate constant for reaction of 1 with all other components of the medium, and ku is the rate constant for reaction of 1 with cells having a number density N in the medium. Since Jurkat cells only take up a small amount of the platinum present in the medium 

Dabrowiak, James C

Di Pasqua, Anthony J

Goodisman, Jerry

Kerwood, Deborah J.

Toms, Bonnie B

English

Using [1H,15N] heteronuclear single quantum coherance (HSQC) NMR and 15N-labeled carboplatin, 1, we show that Jurkat cells affect the rate of disappearance of the HSQC NMR peak in culture medium for this Pt2+ anticancer drug. The decay or disappearance rate constant for 1 in culture medium containing cells is  k1=kc[CO32-]+km+kuN, where kc is the rate constant for reaction of 1 with carbonate in the medium, km is the rate constant for reaction of 1 with all other components of the medium, and ku is the rate constant for reaction of 1 with cells having a number density N in the medium. Since Jurkat cells only take up a small amount of the platinum present in the medium (\u3c1%), the observed disappearance of the HSQC NMR peak for 1 cannot be due to uptake of carboplatin by the cells

Syracuse University Research Facility and Collaborative Environment

Syracuse University SURFACE Chemistry - Faculty Scholarship College of Arts and Sciences 10-2007 Modification of carboplatin by Jurkat cells Anthony J. Di Pasqua Syracuse University Jerry Goodisman Syracuse University Deborah J. Kerwood Syracuse University James C. Dabrowiak Syracuse University Bonnie B. Toms SUNY Upstate Medical University Follow this and additional works at: https://surface.syr.edu/che  Part of the Chemistry Commons Recommended Citation Di Pasqua, Anthony J.; Goodisman, Jerry; Kerwood, Deborah J.; Dabrowiak, James C.; and Toms, Bonnie B., "Modification of carboplatin by Jurkat cells" (2007). Chemistry - Faculty Scholarship. 90. https://surface.syr.edu/che/90 This Article is brought to you for free and open access by the College of Arts and Sciences at SURFACE. It has been accepted for inclusion in Chemistry - Faculty Scholarship by an authorized administrator of SURFACE. For more information, please contact surface@syr.edu. Modiﬁcation of carboplatin by Jurkat cellsAnthony J. Di Pasqua a, Jerry Goodisman a, Deborah J. Kerwood a,Bonnie B. Toms b, James C. Dabrowiak a,*a Department of Chemistry, Syracuse University, 111 College Place, CST, Rm 1-014, Syracuse, NY 13244-4100, United Statesb Department of Pediatrics, Upstate Medical University, State University of New York, 750 East Adams Street, Syracuse, NY 13210, United StatesReceived 25 April 2007; received in revised form 6 June 2007; accepted 7 June 2007Available online 21 June 2007AbstractUsing [1H,15N] heteronuclear single quantum coherance (HSQC) NMR and 15N-labeled carboplatin, 1, we show that Jurkat cellsaﬀect the rate of disappearance of the HSQC NMR peak in culture medium for this Pt2+ anticancer drug. The decay or disappearancerate constant for 1 in culture medium containing cells is k1 ¼ kc½CO23  þ km þ kuN , where kc is the rate constant for reaction of 1 withcarbonate in the medium, km is the rate constant for reaction of 1 with all other components of the medium, and ku is the rate constantfor reaction of 1 with cells having a number density N in the medium. Since Jurkat cells only take up a small amount of the platinumpresent in the medium (<1%), the observed disappearance of the HSQC NMR peak for 1 cannot be due to uptake of carboplatin by thecells. 2007 Elsevier Inc. All rights reserved.Keywords: Pt complex; Cells; NMR1. IntroductionCarboplatin, [Pt(NH3)2(CBDCA-O,O0)], 1, whereCBDCA is cyclobutane-1,1-dicarboxylate, Fig. 1, is aPt2+ anticancer drug in wide clinical use [1]. Using 2D[1H,15N] heteronuclear single quantum coherence (HSQC)NMR, 1D 13C and 1H NMR, and UV–visible spectros-copy, we showed that carboplatin reacts with carbonateion, present in culture media, blood and the cytosol, to pro-duce carbonato carboplatin, cis-[Pt(NH3)2(O-CBDCA)-(CO3)]2, and other carbonato complexes [2,3]. In vitrocytotoxicity studies showed that the carbonato complexesare more toxic to human neuroblastoma (SK-N-SH), renalproximal tubule (HK-2) and Namalwa-luc Burkitt’s lym-phoma cells than is intact 1, suggesting that small amountsof carbonato complexes could form in therapy and be thespecies responsible for the antitumor eﬀects of carboplatin[3]. Earlier we also showed that carbonate reacts with therelated drug, cisplatin, and that one of the complexes isselectively modiﬁed by Jurkat cells, immortalized T lym-phocytes, using an extra-cellular mechanism [4,5].We here use [1H,15N] HSQC NMR and 15N-labeled 1 toshow that the rate of disappearance of carboplatin in themedium is aﬀected not only by substances present in theculture medium but also by an unknown substance(s)released by the cells to the medium. As with previouslyreported experiments with cisplatin [4], no HSQC NMRactive product peaks are observed in the NMR experimentwith carboplatin and Jurkat cells. Since Jurkat cells take uponly a small fraction of the total amount of platinum pres-ent in the medium [3], the disappearance of 1 cannot be dueto simple absorption of the compound by the cells. We alsodemonstrate that the results of the cell/NMR experimentsare independent of the nature of the atmosphere abovethe cells. Rate constants for the disappearance of 1 in thepresence of Jurkat cells in a static atmosphere (closedNMR tube) ﬁt well (R2 = 0.96) with the previouslyreported rate constant for the disappearance of carboplatin0162-0134/$ - see front matter  2007 Elsevier Inc. All rights reserved.doi:10.1016/j.jinorgbio.2007.06.018* Corresponding author. Tel.: +1 315 443 4601; fax: +1 315 443 4070.E-mail address: jcdabrow@syr.edu (J.C. Dabrowiak).www.elsevier.com/locate/jinorgbioJournal of Inorganic Biochemistry 101 (2007) 1438–1441JOURNAL OFInorganicBiochemistryin the presence of 4.5 · 107 cells mL1 kept under standardconditions in a humidiﬁed, 5% CO2 atmosphere in a incu-bator [2].2. Materials and methodsK2PtCl4, H2CBDCA,15NH4Cl and D2O (99.9%) werepurchased from Sigma–Aldrich (St. Louis, MO). Fetalbovine serum (FBS) and RPMI-1640 (15–040) were pur-chased from Mediatech (Herndon, VA). 15N-labeled carbo-platin was prepared as previously described [2]. [1H,15N]HSQC NMR spectra were recorded on a Bruker Avance500 MHz NMR equipped with a 5 mm triple axis probein the earlier described manner [5]. Peak volumes in arbi-trary units, excluding 195Pt satellites, were calculated usingBruker software. Each kinetic run consisted of ten succes-sive NMR measurements (each 60 min in length, ns = 48)for a total duration of 10 h. The 15N chemical shift of 1was referenced externally to 1 M (15NH4)2SO4 in 5% D2Owhich was acidiﬁed to pH 1 by the addition of H2SO4.The 1H chemical shift of 1 was referenced externally to 3-trimethylsilyl propionic acid-d4 sodium salt (TSP) in apH 7.15, 23 mM sodium carbonate buﬀer. Freshly pre-pared labeled carboplatin in water was added to culturemedium (RPMI + 10% FBS, 100 lg/mL streptomycin,100 IU/mL penicillin, and 2.0 mM S-glutamine) containingeither 2.7, 8.2, 21.2 or 27.2 · 107 Jurkat cells per mL, in aﬁnal volume of 920 ll, and spectra were recorded in 5%D2O at 37 C. Jurkat cells used were 97% viable, mea-sured by light microscopy, using a hemacytometer understandard trypan blue conditions [6].3. Results and discussionAs is shown in Fig. 2, 15N-labeled carboplatin exhibits asingle HSQC NMR peak due to the two symmetry-equiva-lent ammonia molecules. The pseudo-ﬁrst order decay con-stant, k1, for the disappearance of carboplatin in culturemedium containing Jurkat cells was determined by ﬁttinga plot of carboplatin concentration (calculated from theHSQC NMR peak intensity) versus time to a line anddividing the slope of the linear ﬁt by its y-intercept. Thevalues obtained for k1 are given in Table 1, with statisticalerrors from the linear ﬁts, and are plotted versus cell den-sity in Fig. 3. Also plotted in Fig. 3 are the previouslyreported k1 values for the disappearance of carboplatin inFig. 2. [1H,15N] HSQC NMR spectrum of 110 lM 15N-carboplatin inculture medium (RPMI + 10% FBS, 100 lg/mL streptomycin, 100 IU/mLpenicillin, and 2.0 mM S-glutamine) containing 2.7 · 107 Jurkat cellsmL1, pH = 7.4, 9.6 h after the addition of carboplatin to the mediumcontaining the cells.Table 1Pseudo-ﬁrst order decay constant, k1, for the disappearance of carboplatinin culture medium containing Jurkat cells determined using [1H,15N]HSQC NMRJurkat cell density (cells mL1) k1 (s1)2.7 · 107 2.28 ± 0.60 · 1068.2 · 107 7.36 ± 1.02 · 10621.2 · 107 13.41 ± 2.94 · 10627.2 · 107 15.41 ± 1.67 · 1060.0E+004.0E-068.0E-061.2E-051.6E-050 4 8 12 16 20 24 28cell density, 107cells mL-1observed rate constant, s-1   Fig. 3. Observed rate constant of the disappearance of carboplatinobtained from HSQC NMR as a function of number of cells per mL.Open diamonds are k1 values already reported by us [2]. The best ﬁt line,R2 = 0.96, is k1 = (1.94 · 106 + 5.14 · 1014 mL cell1N) s1, so thatku = 5.14 · 1014 mL cell1 s1.PtH3NH3NOOCCOOH2CCH2CH 21Fig. 1. Structure of carboplatin.A.J. Di Pasqua et al. / Journal of Inorganic Biochemistry 101 (2007) 1438–1441 1439culture medium (no cells) and in culture medium contain-ing 4.5 · 107 cells mL1 kept under standard conditionsin a humidiﬁed 5% CO2 atmosphere in an incubator [2].In the previously described cell experiment [2], aliquots ofcells plus medium were removed from the culture ﬂask,cells were separated from the medium by centrifugation,5% D2O was added to the medium and NMR data was col-lected for 0.5 h. As can be seen in Fig. 3, all the data pointsare consistent with the best ﬁt (R2 = 0.96) line k1s = 1.94 ·106 + 5.14 · 1014 (mL cell1)N, demonstrating that cellstudies will yield the same results regardless if the reactionwith drug is done in a 5 mm capped NMR tube or in ahumidiﬁed, 5% CO2 incubator.A striking feature of the reaction of 1 in these studies isthat no new HSQC NMR peaks associated with productsare observed. As we, as well as Sadler and coworkers, haveshown [2,7–9], reaction of 1 with nucleophiles lifts theequivalency of the two ammonia molecules to producenew peaks, the positions of which are sensitive to the nat-ure of the ligands trans to the ammonia molecules. Sulfurin the form of a thiol (cysteine) or thioether (methionine)is the most potent nucleophile for Pt2+ present in culturemedia. While a sulfur ligand trans to an ammonia moleculewould make the latter vulnerable to a substitution reactiondue to the trans eﬀect, these complexes, with N trans to S,are suﬃciently stable to be easily detected at 40 ppm(N) using HSQC NMR [4,7–11]. No products of this typeor of any other species were observed in the reaction.The prospect that the disappearance of the HSQC NMRpeak for 1 in the presence of cells is due to absorption ofcarboplatin by the cells is highly unlikely. As we earliershowed, Jurkat cells under conditions similar to those usedhere remove platinum species from solution at a rate whichis only 16.6 ± 4.2 · 1018 mol Pt h1 cell1 [3]. This rate ofplatinum removal by cells is too slow to show an observa-ble eﬀect on the total pool of 1 in the medium above thecells during the time course of these experiments.In the presence of culture medium without cells, the con-centration of carboplatin [1] should obeyd½1dt¼ kc½1½CO23   km½1where kc is the rate constant for reaction of 1 with carbon-ate in the medium, a second order process, which behaveslike a pseudo-ﬁrst order reaction because [CO23 ] isconstant under the reaction conditions used [2]. The quan-tity km is the rate constant for reaction of 1 with othersubstances in the medium, which is assumed to be a ﬁrst or-der process. For example, earlier studies showed that FBS,a suite of proteins needed for cell growth, is also a signiﬁ-cant factor causing the disappearance of 1 in culture media[2].If cells, or some substance released by them, can modifycarboplatin so that it does not give an NMR signal, a termku[1]N should be added to the equation for d[1]/dt, whereN is the density of cells in the medium. The solution to theresulting equation is an exponential decay,½1 ¼ ½10 expfðkc½CO23  þ km þ kuNÞtg ¼ ½10 expfk1tgwhere the eﬀective decay constant k1 ¼ kc½CO23  þ kmþkuN . The observed values of k1, shown in Fig. 3, dependlinearly on cell density. The y-intercept, 1.94 ± 0.76 ·106 s1 represents kc½CO23  þ km; from the slope, we ob-tain ku = 5.14 ± 0.52 · 1014 mL cell1 s1. When the den-sity of cells is P8.2 · 107 cells mL1, the reaction ofcarboplatin with cells is more important than reaction ofdrug with carbonate or other substances in the culturemedium. Since, as noted, direct removal of 1 by cells isunimportant, the reaction must involve a substance re-leased by the cells.Earlier studies by us using cisplatin [4,5] showed thatJurkat cells can rapidly modify the mono-carbonato com-plex, cis-[Pt(NH3)2(CO3)Cl], converting it into a sub-stance that is not capable of binding to the cells and isundetectable using HSQC NMR. Moreover, the amountof modiﬁcation of the mono-carbonato complex is depen-dent on the number of cells and recent studies by our group(Centerwall et al., personal communication) show that theplatinum-modifying substance is released to the culturemedium above the cells. Unlike the relatively slow decayrate observed for carboplatin in the presence of cells inthese experiments, the rate of modiﬁcation of the cisplatinmono-carbonato complex by Jurkat cells is extremelyrapid, occurring within 0.6 h of the addition of drug tothe culture medium above the cells [4,5]. It seems likely thatboth drugs are exposed to the same cell-released modifyingsubstance, but since carboplatin has a bidentate chelatering which blocks nucleophiles from attacking the axialsites of the compound, it is much less susceptible to chem-ical modiﬁcation than is the cisplatin mono-carbonato spe-cies. Clearly, additional work is needed to determine thenature of the species released by the cells and productsformed in the reaction.4. ConclusionJurkat cells release a substance to the culture mediumthat reacts with carboplatin, converting it into a substancethat, under the conditions of the experiment, is notdetectable using HSQC NMR. This disappearance fromHSQC NMR is identical to that observed for a mono-car-bonato complex of cisplatin except that, due to structuraldiﬀerences between cisplatin and carboplatin, the rate ofreaction with carboplatin is much slower than withcisplatin.References[1] T. Boulikas, M. Vougiouka, Oncol. Rep. 10 (2003) 1663–1682.[2] A.J. Di Pasqua, J. Goodisman, D.J. Kerwood, B.B. Toms,R.L. Dubowy, J.C. Dabrowiak, Chem. Res. Toxicol. 19 (2006) 139–149.[3] A.J. Di Pasqua, J. Goodisman, D.J. Kerwood, B.B. Toms, R.L.Dubowy, J.C. Dabrowiak, Chem. Res. Toxicol. 20 (2007) 896–904.1440 A.J. Di Pasqua et al. / Journal of Inorganic Biochemistry 101 (2007) 1438–1441[4] C.R. Centerwall, K.A. Tacka, D.J. Kerwood, J. Goodisman, J.B.B.Toms, R.L. Dubowy, J.C. Dabrowiak, Mol. Pharmacol. 70 (2006)348–355.[5] K.A. Tacka, D. Szalda, A.-K. Souid, J. Goodisman, J.C. Dabrowiak,Chem. Res. Toxicol. 17 (2004) 1434–1444.[6] D.C. Allison, P. Riddolpho, J. Histochem. 28 (1980) 700–703.[7] K.J. Barnham, M.I. Djuran, P.S. Murdoch, J.D. Ranford, P.J.Sadler, Inorg. Chem. 35 (1996) 1065–1072.[8] K.J. Barnham, U. Frey, P.P. Murdoch, J.D. Ranford, P.J. Sadler, J.Am. Chem. Soc. 116 (1994) 11175–11176.[9] J.D. Ranford, P.J. Sadler, K. Balmanno, D.R. Newell, Magn. Reson.Chem. 29 (1991) S125–S129.[10] M. El-Khateeb, T.G. Appleton, L.R. Gahan, B.G. Charles, S.J.Berners-Price, A. Bolton, J. Inorg. Biochem. 77 (1999) 13–21.[11] S.J. Berners-Price, L. Ronconi, P.J. Sadler, Prog. Nucl. Mag. Res. Sp.49 (2006) 65–98.A.J. Di Pasqua et al. / Journal of Inorganic Biochemistry 101 (2007) 1438–1441 1441

Modification of carboplatin by Jurkat cells

Syracuse University SURFACE Chemistry - Faculty Scholarship College of Arts and Sciences 6-21-2007 Modification of Carboplatin by Jurkat Cells Anthony J. Di Pasqua Syracuse University Jerry Goodisman Syracuse University Deborah J. Kerwood Syracuse University Bonnie B. Toms Upstate Medical University, State University of New York James C. Dabrowiak Syracuse University Follow this and additional works at: https://surface.syr.edu/che  Part of the Chemistry Commons Recommended Citation Di Pasqua, Anthony J.; Goodisman, Jerry; Kerwood, Deborah J.; Toms, Bonnie B.; and Dabrowiak, James C., "Modification of Carboplatin by Jurkat Cells" (2007). Chemistry - Faculty Scholarship. 30. https://surface.syr.edu/che/30 This Article is brought to you for free and open access by the College of Arts and Sciences at SURFACE. It has been accepted for inclusion in Chemistry - Faculty Scholarship by an authorized administrator of SURFACE. For more information, please contact surface@syr.edu. Modiﬁcation of carboplatin by Jurkat cellsAnthony J. Di Pasqua a, Jerry Goodisman a, Deborah J. Kerwood a,Bonnie B. Toms b, James C. Dabrowiak a,*a Department of Chemistry, Syracuse University, 111 College Place, CST, Rm 1-014, Syracuse, NY 13244-4100, United Statesb Department of Pediatrics, Upstate Medical University, State University of New York, 750 East Adams Street, Syracuse, NY 13210, United StatesReceived 25 April 2007; received in revised form 6 June 2007; accepted 7 June 2007Available online 21 June 2007AbstractUsing [1H,15N] heteronuclear single quantum coherance (HSQC) NMR and 15N-labeled carboplatin, 1, we show that Jurkat cellsaﬀect the rate of disappearance of the HSQC NMR peak in culture medium for this Pt2+ anticancer drug. The decay or disappearancerate constant for 1 in culture medium containing cells is k1 ¼ kc½CO23  þ km þ kuN , where kc is the rate constant for reaction of 1 withcarbonate in the medium, km is the rate constant for reaction of 1 with all other components of the medium, and ku is the rate constantfor reaction of 1 with cells having a number density N in the medium. Since Jurkat cells only take up a small amount of the platinumpresent in the medium (<1%), the observed disappearance of the HSQC NMR peak for 1 cannot be due to uptake of carboplatin by thecells. 2007 Elsevier Inc. All rights reserved.Keywords: Pt complex; Cells; NMR1. IntroductionCarboplatin, [Pt(NH3)2(CBDCA-O,O0)], 1, whereCBDCA is cyclobutane-1,1-dicarboxylate, Fig. 1, is aPt2+ anticancer drug in wide clinical use [1]. Using 2D[1H,15N] heteronuclear single quantum coherence (HSQC)NMR, 1D 13C and 1H NMR, and UV–visible spectros-copy, we showed that carboplatin reacts with carbonateion, present in culture media, blood and the cytosol, to pro-duce carbonato carboplatin, cis-[Pt(NH3)2(O-CBDCA)-(CO3)]2, and other carbonato complexes [2,3]. In vitrocytotoxicity studies showed that the carbonato complexesare more toxic to human neuroblastoma (SK-N-SH), renalproximal tubule (HK-2) and Namalwa-luc Burkitt’s lym-phoma cells than is intact 1, suggesting that small amountsof carbonato complexes could form in therapy and be thespecies responsible for the antitumor eﬀects of carboplatin[3]. Earlier we also showed that carbonate reacts with therelated drug, cisplatin, and that one of the complexes isselectively modiﬁed by Jurkat cells, immortalized T lym-phocytes, using an extra-cellular mechanism [4,5].We here use [1H,15N] HSQC NMR and 15N-labeled 1 toshow that the rate of disappearance of carboplatin in themedium is aﬀected not only by substances present in theculture medium but also by an unknown substance(s)released by the cells to the medium. As with previouslyreported experiments with cisplatin [4], no HSQC NMRactive product peaks are observed in the NMR experimentwith carboplatin and Jurkat cells. Since Jurkat cells take uponly a small fraction of the total amount of platinum pres-ent in the medium [3], the disappearance of 1 cannot be dueto simple absorption of the compound by the cells. We alsodemonstrate that the results of the cell/NMR experimentsare independent of the nature of the atmosphere abovethe cells. Rate constants for the disappearance of 1 in thepresence of Jurkat cells in a static atmosphere (closedNMR tube) ﬁt well (R2 = 0.96) with the previouslyreported rate constant for the disappearance of carboplatin0162-0134/$ - see front matter  2007 Elsevier Inc. All rights reserved.doi:10.1016/j.jinorgbio.2007.06.018* Corresponding author. Tel.: +1 315 443 4601; fax: +1 315 443 4070.E-mail address: jcdabrow@syr.edu (J.C. Dabrowiak).www.elsevier.com/locate/jinorgbioJournal of Inorganic Biochemistry 101 (2007) 1438–1441JOURNAL OFInorganicBiochemistryin the presence of 4.5 · 107 cells mL1 kept under standardconditions in a humidiﬁed, 5% CO2 atmosphere in a incu-bator [2].2. Materials and methodsK2PtCl4, H2CBDCA,15NH4Cl and D2O (99.9%) werepurchased from Sigma–Aldrich (St. Louis, MO). Fetalbovine serum (FBS) and RPMI-1640 (15–040) were pur-chased from Mediatech (Herndon, VA). 15N-labeled carbo-platin was prepared as previously described [2]. [1H,15N]HSQC NMR spectra were recorded on a Bruker Avance500 MHz NMR equipped with a 5 mm triple axis probein the earlier described manner [5]. Peak volumes in arbi-trary units, excluding 195Pt satellites, were calculated usingBruker software. Each kinetic run consisted of ten succes-sive NMR measurements (each 60 min in length, ns = 48)for a total duration of 10 h. The 15N chemical shift of 1was referenced externally to 1 M (15NH4)2SO4 in 5% D2Owhich was acidiﬁed to pH 1 by the addition of H2SO4.The 1H chemical shift of 1 was referenced externally to 3-trimethylsilyl propionic acid-d4 sodium salt (TSP) in apH 7.15, 23 mM sodium carbonate buﬀer. Freshly pre-pared labeled carboplatin in water was added to culturemedium (RPMI + 10% FBS, 100 lg/mL streptomycin,100 IU/mL penicillin, and 2.0 mM S-glutamine) containingeither 2.7, 8.2, 21.2 or 27.2 · 107 Jurkat cells per mL, in aﬁnal volume of 920 ll, and spectra were recorded in 5%D2O at 37 C. Jurkat cells used were 97% viable, mea-sured by light microscopy, using a hemacytometer understandard trypan blue conditions [6].3. Results and discussionAs is shown in Fig. 2, 15N-labeled carboplatin exhibits asingle HSQC NMR peak due to the two symmetry-equiva-lent ammonia molecules. The pseudo-ﬁrst order decay con-stant, k1, for the disappearance of carboplatin in culturemedium containing Jurkat cells was determined by ﬁttinga plot of carboplatin concentration (calculated from theHSQC NMR peak intensity) versus time to a line anddividing the slope of the linear ﬁt by its y-intercept. Thevalues obtained for k1 are given in Table 1, with statisticalerrors from the linear ﬁts, and are plotted versus cell den-sity in Fig. 3. Also plotted in Fig. 3 are the previouslyreported k1 values for the disappearance of carboplatin inFig. 2. [1H,15N] HSQC NMR spectrum of 110 lM 15N-carboplatin inculture medium (RPMI + 10% FBS, 100 lg/mL streptomycin, 100 IU/mLpenicillin, and 2.0 mM S-glutamine) containing 2.7 · 107 Jurkat cellsmL1, pH = 7.4, 9.6 h after the addition of carboplatin to the mediumcontaining the cells.Table 1Pseudo-ﬁrst order decay constant, k1, for the disappearance of carboplatinin culture medium containing Jurkat cells determined using [1H,15N]HSQC NMRJurkat cell density (cells mL1) k1 (s1)2.7 · 107 2.28 ± 0.60 · 1068.2 · 107 7.36 ± 1.02 · 10621.2 · 107 13.41 ± 2.94 · 10627.2 · 107 15.41 ± 1.67 · 1060.0E+004.0E-068.0E-061.2E-051.6E-050 4 8 12 16 20 24 28cell density, 107cells mL-1observed rate constant, s-1   Fig. 3. Observed rate constant of the disappearance of carboplatinobtained from HSQC NMR as a function of number of cells per mL.Open diamonds are k1 values already reported by us [2]. The best ﬁt line,R2 = 0.96, is k1 = (1.94 · 106 + 5.14 · 1014 mL cell1N) s1, so thatku = 5.14 · 1014 mL cell1 s1.PtH3NH3NOOCCOOH2CCH2CH 21Fig. 1. Structure of carboplatin.A.J. Di Pasqua et al. / Journal of Inorganic Biochemistry 101 (2007) 1438–1441 1439culture medium (no cells) and in culture medium contain-ing 4.5 · 107 cells mL1 kept under standard conditionsin a humidiﬁed 5% CO2 atmosphere in an incubator [2].In the previously described cell experiment [2], aliquots ofcells plus medium were removed from the culture ﬂask,cells were separated from the medium by centrifugation,5% D2O was added to the medium and NMR data was col-lected for 0.5 h. As can be seen in Fig. 3, all the data pointsare consistent with the best ﬁt (R2 = 0.96) line k1s = 1.94 ·106 + 5.14 · 1014 (mL cell1)N, demonstrating that cellstudies will yield the same results regardless if the reactionwith drug is done in a 5 mm capped NMR tube or in ahumidiﬁed, 5% CO2 incubator.A striking feature of the reaction of 1 in these studies isthat no new HSQC NMR peaks associated with productsare observed. As we, as well as Sadler and coworkers, haveshown [2,7–9], reaction of 1 with nucleophiles lifts theequivalency of the two ammonia molecules to producenew peaks, the positions of which are sensitive to the nat-ure of the ligands trans to the ammonia molecules. Sulfurin the form of a thiol (cysteine) or thioether (methionine)is the most potent nucleophile for Pt2+ present in culturemedia. While a sulfur ligand trans to an ammonia moleculewould make the latter vulnerable to a substitution reactiondue to the trans eﬀect, these complexes, with N trans to S,are suﬃciently stable to be easily detected at 40 ppm(N) using HSQC NMR [4,7–11]. No products of this typeor of any other species were observed in the reaction.The prospect that the disappearance of the HSQC NMRpeak for 1 in the presence of cells is due to absorption ofcarboplatin by the cells is highly unlikely. As we earliershowed, Jurkat cells under conditions similar to those usedhere remove platinum species from solution at a rate whichis only 16.6 ± 4.2 · 1018 mol Pt h1 cell1 [3]. This rate ofplatinum removal by cells is too slow to show an observa-ble eﬀect on the total pool of 1 in the medium above thecells during the time course of these experiments.In the presence of culture medium without cells, the con-centration of carboplatin [1] should obeyd½1dt¼ kc½1½CO23   km½1where kc is the rate constant for reaction of 1 with carbon-ate in the medium, a second order process, which behaveslike a pseudo-ﬁrst order reaction because [CO23 ] isconstant under the reaction conditions used [2]. The quan-tity km is the rate constant for reaction of 1 with othersubstances in the medium, which is assumed to be a ﬁrst or-der process. For example, earlier studies showed that FBS,a suite of proteins needed for cell growth, is also a signiﬁ-cant factor causing the disappearance of 1 in culture media[2].If cells, or some substance released by them, can modifycarboplatin so that it does not give an NMR signal, a termku[1]N should be added to the equation for d[1]/dt, whereN is the density of cells in the medium. The solution to theresulting equation is an exponential decay,½1 ¼ ½10 expfðkc½CO23  þ km þ kuNÞtg ¼ ½10 expfk1tgwhere the eﬀective decay constant k1 ¼ kc½CO23  þ kmþkuN . The observed values of k1, shown in Fig. 3, dependlinearly on cell density. The y-intercept, 1.94 ± 0.76 ·106 s1 represents kc½CO23  þ km; from the slope, we ob-tain ku = 5.14 ± 0.52 · 1014 mL cell1 s1. When the den-sity of cells is P8.2 · 107 cells mL1, the reaction ofcarboplatin with cells is more important than reaction ofdrug with carbonate or other substances in the culturemedium. Since, as noted, direct removal of 1 by cells isunimportant, the reaction must involve a substance re-leased by the cells.Earlier studies by us using cisplatin [4,5] showed thatJurkat cells can rapidly modify the mono-carbonato com-plex, cis-[Pt(NH3)2(CO3)Cl], converting it into a sub-stance that is not capable of binding to the cells and isundetectable using HSQC NMR. Moreover, the amountof modiﬁcation of the mono-carbonato complex is depen-dent on the number of cells and recent studies by our group(Centerwall et al., personal communication) show that theplatinum-modifying substance is released to the culturemedium above the cells. Unlike the relatively slow decayrate observed for carboplatin in the presence of cells inthese experiments, the rate of modiﬁcation of the cisplatinmono-carbonato complex by Jurkat cells is extremelyrapid, occurring within 0.6 h of the addition of drug tothe culture medium above the cells [4,5]. It seems likely thatboth drugs are exposed to the same cell-released modifyingsubstance, but since carboplatin has a bidentate chelatering which blocks nucleophiles from attacking the axialsites of the compound, it is much less susceptible to chem-ical modiﬁcation than is the cisplatin mono-carbonato spe-cies. Clearly, additional work is needed to determine thenature of the species released by the cells and productsformed in the reaction.4. ConclusionJurkat cells release a substance to the culture mediumthat reacts with carboplatin, converting it into a substancethat, under the conditions of the experiment, is notdetectable using HSQC NMR. This disappearance fromHSQC NMR is identical to that observed for a mono-car-bonato complex of cisplatin except that, due to structuraldiﬀerences between cisplatin and carboplatin, the rate ofreaction with carboplatin is much slower than withcisplatin.References[1] T. Boulikas, M. Vougiouka, Oncol. Rep. 10 (2003) 1663–1682.[2] A.J. Di Pasqua, J. Goodisman, D.J. Kerwood, B.B. Toms,R.L. Dubowy, J.C. Dabrowiak, Chem. Res. Toxicol. 19 (2006) 139–149.[3] A.J. Di Pasqua, J. Goodisman, D.J. Kerwood, B.B. Toms, R.L.Dubowy, J.C. Dabrowiak, Chem. Res. Toxicol. 20 (2007) 896–904.1440 A.J. Di Pasqua et al. / Journal of Inorganic Biochemistry 101 (2007) 1438–1441[4] C.R. Centerwall, K.A. Tacka, D.J. Kerwood, J. Goodisman, J.B.B.Toms, R.L. Dubowy, J.C. Dabrowiak, Mol. Pharmacol. 70 (2006)348–355.[5] K.A. Tacka, D. Szalda, A.-K. Souid, J. Goodisman, J.C. Dabrowiak,Chem. Res. Toxicol. 17 (2004) 1434–1444.[6] D.C. Allison, P. Riddolpho, J. Histochem. 28 (1980) 700–703.[7] K.J. Barnham, M.I. Djuran, P.S. Murdoch, J.D. Ranford, P.J.Sadler, Inorg. Chem. 35 (1996) 1065–1072.[8] K.J. Barnham, U. Frey, P.P. Murdoch, J.D. Ranford, P.J. Sadler, J.Am. Chem. Soc. 116 (1994) 11175–11176.[9] J.D. Ranford, P.J. Sadler, K. Balmanno, D.R. Newell, Magn. Reson.Chem. 29 (1991) S125–S129.[10] M. El-Khateeb, T.G. Appleton, L.R. Gahan, B.G. Charles, S.J.Berners-Price, A. Bolton, J. Inorg. Biochem. 77 (1999) 13–21.[11] S.J. Berners-Price, L. Ronconi, P.J. Sadler, Prog. Nucl. Mag. Res. Sp.49 (2006) 65–98.A.J. Di Pasqua et al. / Journal of Inorganic Biochemistry 101 (2007) 1438–1441 1441

Modification of Carboplatin by Jurkat Cells

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Modification of Carboplatin by Jurkat Cells

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