Abstract C171: TriplatinNC, a nucleolar-targeting platinum agent. Heparan sulfate-mediated cell entry, localization, and apoptosis.

Abstract

TriplatinNC, a “non-covalent” derivative of the phase II clinical drug BBR3464, exhibits a distinct mode of DNA binding mediated through “phosphate clamps”, which display structural analogy to the “polyarginine” (arginine fork) DNA/RNA recognition motif. TriplatinNC shows an improved pharmacokinetic profile and increased cellular accumulation compared to BBR3464. Furthering the “polyarginine” analogy, the cellular accumulation of TriplatinNC, unlike cisplatin or oxaliplatin, is dependent upon the presence of cell surface glycosaminoglycans (GAGs), shown through comparison of platinum level accumulation in wild-type Chinese Hamster Ovary (CHO) cell lines to those lacking heparan sulfate (CHO-pgsD-677) or both heparan sulfate and chondroitin sulfate (CHO-pgsA-745). The cellular localization of TriplatinNC and cisplatin was visualized in confocal microscopy experiments utilizing the fluorescently-tagged derivatives, TriplatinNC-NBD or cisplatin-NBD. TriplatinNC, but not cisplatin, shows distinct localization to the nucleolar region of human colon and ovarian carcinoma cell lines, HCT116 and A2780, respectively. It was hypothesized that this pattern of localization, combined with nucleic acid affinity, would allow TriplatinNC to disrupt rRNA synthesis, the primary function within the nucleolus. Using 32P-metabolic labeling to monitor the rate of pre-rRNA transcript formation after cellular treatment, it was observed that both 47S pre-RNA and processed rRNAs, 32S, 28S, and 18S levels decreased dramatically compared to the untreated control in a dose-dependent manner. While the localization and abundance of nucleolar proteins, RNA pol I and UBTF, were unaffected by treatments with TriplatinNC at early timepoints, levels of the clinical relevant proliferative marker Ki-67, decreased markedly within one hour post-treatment. The morphological characteristics of apoptosis, including reduction in cell size, membrane blebbing, and cytosolic vacuolization, become visible 10hrs after drug treatment. This apoptotic process is not dependent on p53, as western blot analysis showed cleavage of procaspases 8, 9, and 3, followed by Parp-1 in HCT116 p53+/+ and p53−/− cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C171.