Heparan Sulfate Proteoglycan-Mediated Entry Pathway for Charged Tri-Platinum Compounds: Differential Cellular Accumulation Mechanisms for Platinum

We examined the mechanism of accumulation of charged polynuclear platinum complexes (PPCs) based on analogy of polyarginine interactions with the cell surface heparan sulfate proteoglycan (HSPG) family of protein-linked glycosoaminoglycan polysaccharides (GAGs). GAGS such as heparan sulfate (HS) and chondroitin sulfate (CS) mediate the cellular entry of many charged molecules. Fluorescence microscopy and flow cytometry showed that PPCs, but not the neutral cisplatin or oxaliplatin, blocked the cellular entry of TAMRA-R₉ (a nonarginine peptide, R₉) coupled to the TAMRA fluorescent label 5-(and 6-)­carboxytetramethylrhodamine) in Chinese hamster ovary (CHO), human colon carcinoma (HCT116), and osteosarcoma (SAOS-2) cells. Furthermore, detection of platinum accumulation in wt CHO, mutant CHO-pgsD-677 (lacking HS), and CHO-pgsA (lacking HS/CS) cells confirms that HSPG-mediated interactions are an important mechanism for PPC internalization but not so for uncharged cisplatin and oxaliplatin. Endocytosis inhibitor studies show that macropinocytosis, a mechanism of cell entry for heparan sulfate GAGs and arginine-rich peptides, is important in the cellular accumulation of noncovalent TriplatinNC and, to a lesser degree, the covalently binding BBR3464. Clathrin-mediated endocytosis, however, was not involved in either case. Overall, the results suggest a new proteoglycan-mediated mechanism for cellular accumulation of PPCs not shared by cisplatin or oxaliplatin. The results have significant implications for the rational design of platinum antitumor drugs with distinct biological profiles in comparison to those of the clinically used agents as well as expanding the chemotypes for HS proteoglycan-dependent receptors