Role of Carbonate in the Cytotoxicity of Carboplatin

Abstract

Carboplatin, [Pt(NH3)2(CBDCA-O,O')], 1, where CBDCA is cyclobutane-1,1-dicarboxylate, is used against ovarian, lung, and other types of cancer. We recently showed (Di Pasqua et al. (2006) Chem. Res. Toxicol. 19, 139-149) that carboplatin reacts with carbonate under conditions that simulate therapy to produce carbonato carboplatin, cis-[Pt(NH3)2(O-CBDCA)(CO3)]2-, 2. We use 13C and 1H NMR and UV-visible absorption spectroscopy to show that solutions containing carboplatin that have been aged in carbonate buffer under various conditions contain 1, 2, and other compounds. We then show that aging carboplatin in carbonate produces compounds that are more toxic to human neuroblastoma (SK-N-SH), proximal renal tubule (HK-2) and Namalwa-luc Burkitt's lymphoma (BL) cells than carboplatin alone. Moreover, increasing the aging time increases the cytotoxicity of the platinum solutions as measured by the increase in cell death. Although HK-2 cells experience a large loss in survival upon exposure to carbonato forms of the drug, they have the highest values of IC50 of the three cell lines studied, so that HK-2 cells remain the most resistant to the toxic effects of the carbonato forms in the culture medium. This is consistent with the well-known low renal toxicity observed for carboplatin in therapy. The uptake rates for normal Jurkat cells (NJ) and cisplatin-resistant Jurkat cells (RJ), measured by inductively coupled plasma mass spectrometry (ICP-MS), are 16.6 +/- 4.2 and 12.3 +/- 4.8 amol of Pt h-1 cell-1, respectively, when exposed to carboplatin alone. However, when these cells are exposed to carboplatin that has been aged in carbonate media, normal Jurkat cells strongly bind/take up Pt at a rate of 14.5 +/- 4.1 amol of Pt h-1 cell-1, while resistant cells strongly bind/take up 5.1 +/- 3.3 amol of Pt h-1 cell-1. Collectively, these studies show that carboplatin carbonato species may play a major role in the cytotoxicity and uptake of carboplatin by cells.

Influence of Carbonate on the Binding of Carboplatin to DNA

“…It was theorized that these ring-opened species could be the active agents responsible for carboplatin's anticancer activity in the body, and that carbonate ion is the attacking nucleophile responsible for activating the drug in vivo. This was supported by Di Pasqua's 2007 finding that carbonate-aged carboplatin is more cytotoxic to human neuroblastoma, proximal renal tube, and Namalwa-luc Burkitt's lymphoma cells than the native drug (24). It was also seen that Jurkat cells produce an unidentified extracellular substance which modifies carboplatin (25), similar to the defense mechanism against cisplatin reported by Dabrowiak and coworkers (20).…”

“…We followed the carbonate aging procedure outlined by Di Pasqua (23,24), providing the full variety of carbonatocarboplatin species thought to develop under physiological conditions. By carrying out DNA binding in a carbonate-rich solution, we modeled the environment of cellular DNA in vivo.…”

Adsorption of the PtII Anticancer Drug Carboplatin by Mesoporous Silica

“…without 1 as a baseline for 30 h. Difference spectra were obtained by subtracting the spectrum immediately after addition of 1 to H 2 O (t ¼ 0 spectrum) from spectra taken at various times. Each difference spectrum was fitted to a sum of Gaussians using PeakFit (v. 4) [17] [25].…”

Modification of carboplatin by Jurkat cells

“…Using 2D [ 1 H, 15 N] heteronuclear single quantum coherence (HSQC) NMR, 1D 13 C and 1 H 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(NH 3 ) 2 (O-CBDCA)-(CO 3 )] À2 , and other carbonato complexes [2,3]. In vitro cytotoxicity studies showed that the carbonato complexes are more toxic to human neuroblastoma (SK-N-SH), renal proximal tubule (HK-2) and Namalwa-luc Burkitt's lymphoma cells than is intact 1, suggesting that small amounts of carbonato complexes could form in therapy and be the species responsible for the antitumor effects of carboplatin [3]. Earlier we also 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].…”

“…As with previously reported experiments with cisplatin [4], no HSQC NMR active product peaks are observed in the NMR experiment with carboplatin and Jurkat cells. Since Jurkat cells take up only a small fraction of the total amount of platinum present in the medium [3], the disappearance of 1 cannot be due to simple absorption of the compound by the cells. We also demonstrate that the results of the cell/NMR experiments are independent of the nature of the atmosphere above the cells.…”