Abstract 3501: Characterisation of cisplatin-DNA adducts formed in the presence of sodium thiosulfate

Abstract

Abstract Sodium thiosulfate (STS) is currently in a phase III clinical trial to investigate its efficacy in reducing ototoxicity in patients receiving cisplatin for standard risk hepatoblastoma. In animal studies STS has shown protection against ototoxicity, although the mechanism through which this occurs is unknown. It is unclear to what extent STS enters cells, but there is evidence linking uptake with the sulfate transporters. Intracellular STS has the potential to directly affect platination of DNA. The aim of this study was to test the hypothesis that STS can alter the types of DNA adducts formed by cisplatin and to compare its effects on cisplatin sensitivity and adduct levels in cancer cell lines. Purified DNA was reacted with cisplatin alone or in combination with STS (2mg/ml) and enzymatically hydrolysed to nucleotides. Equimolar (1mM) deoxyguanosine monophosphate (dGMP) and cisplatin were reacted for 24h followed by reaction with STS for a further 24h. DNA extracted from human tumour cell lines (833K, A2780, LoVo and MorCPR) was hydrolysed in nitric acid. Anion-exchange chromatography was used in conjunction with atomic absorption spectrometry for dGMP reactions and inductively-coupled plasma mass spectrometry (ICP-MS) for calf thymus and cellular DNA to detect Pt species. Growth inhibition was measured using the sulphorhodamine B assay. MonoQ chromatographic analysis of adducts formed on purified DNA confirmed the formation of the major 1, 2-intrastrand cross-links (ApG and GpG). Total adduct levels were decreased approximately 10-fold in the presence of STS. In addition a novel Pt-containing product was detected, eluting later than the previously characterized adducts and accounting for 25% of total adduct formation. To test the hypothesis that the product was a DNA-Pt-thiosulfate cross-link, cis-Pt [(NH3)2Cl.dGMP)] was reacted with STS. A major Pt-containing product with the same chromatographic properties as the product identified in DNA was detected. Cisplatin, alone or in combination with STS (2mg/ml) was added to the cell lines and removed after 6h. In additional experiments STS was added immediately after or 6h after cisplatin removal. Total incubation time was 72h. Co-incubation of cisplatin and STS led to increases in IC50 of 1.4 to 50.3µM, 2.2 to 45.5µM, 2.7 to 60.2µM and 29.4 to >500µM in 833K, A2780, LoVo and MorCPR cells respectively, with associated 10-15 fold decreases in adduct levels observed. Delayed additions of STS had no significant effect on growth inhibition or overall adduct levels. Anion-exchange chromatography with ICP-MS demonstrated that STS has the potential to modify platination of DNA. However, analysis of four cell lines showed no evidence for STS affecting levels of preformed DNA adducts in cells, supporting the hypothesis that the mechanism of otoprotection is through covalent binding and inactivation of cisplatin, without affecting the platination of DNA in tumour cells. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3501.