Abstract
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 Pt 2+ anticancer drug. The decay or disappearance rate constant for 1 in culture medium containing cells is k 1 = k c [ CO 3 2 - ] + k m + k u N , where k c is the rate constant for reaction of 1 with carbonate in the medium, k m is the rate constant for reaction of 1 with all other components of the medium, and k u 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 (<1%), the observed disappearance of the HSQC NMR peak for 1 cannot be due to uptake of carboplatin by the cells.
Highlights
Carboplatin, [Pt(NH3)2(CBDCA-O,O0)], 1, where CBDCA is cyclobutane-1,1-dicarboxylate, Fig. 1, is a Pt2+ 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 spectroscopy, we showed that carboplatin reacts with carbonate ion, present in culture media, blood and the cytosol, to produce carbonato carboplatin, cis-[Pt(NH3)2(O-CBDCA)(CO3)]À2, and other carbonato complexes [2,3]
Prepared labeled carboplatin in water was added to culture medium (RPMI + 10% Fetal bovine serum (FBS), 100 lg/mL streptomycin, 100 IU/mL penicillin, and 2.0 mM S-glutamine) containing either 2.7, 8.2, 21.2 or 27.2 · 107 Jurkat cells per mL, in a final volume of 920 ll, and spectra were recorded in 5% D2O at 37 °C
Summary
Carboplatin, [Pt(NH3)2(CBDCA-O,O0)], 1, where CBDCA is cyclobutane-1,1-dicarboxylate, Fig. 1, is a Pt2+ anticancer drug in wide clinical use [1]. Earlier we showed that carbonate reacts with the related drug, cisplatin, and that one of the complexes is selectively modified by Jurkat cells, immortalized T lymphocytes, using an extra-cellular mechanism [4,5]. We here use [1H,15N] HSQC NMR and 15N-labeled 1 to show that the rate of disappearance of carboplatin in the medium is affected by substances present in the culture medium and by an unknown substance(s) released by the cells to the medium. Rate constants for the disappearance of 1 in the presence of Jurkat cells in a static atmosphere (closed NMR tube) fit well (R2 = 0.96) with the previously reported rate constant for the disappearance of carboplatin. In the presence of 4.5 · 107 cells mLÀ1 kept under standard conditions in a humidified, 5% CO2 atmosphere in a incubator [2]
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