Nucleolar Targeting by Platinum: p53-Independent Apoptosis Follows rRNA Inhibition, Cell-Cycle Arrest, and DNA Compaction

Abstract

TriplatinNC is a highly positively charged, substitution-inert derivative of the phase II clinical anticancer drug, BBR3464. Such substitution-inert complexes form a distinct subset of polynuclear platinum complexes (PPCs) interacting with DNA and other biomolecules through noncovalent interactions. Rapid cellular entry is facilitated via interaction with cell surface glycosoaminoglycans and is a mechanism unique to PPCs. Nanoscale secondary ion mass spectrometry (nanoSIMS) showed rapid distribution within cytoplasmic and nucleolar compartments, but not the nucleus. In this article, the downstream effects of nucleolar localization are described. In human colon carcinoma cells, HCT116, the production rate of 47S rRNA precursor transcripts was dramatically reduced as an early event after drug treatment. Transcriptional inhibition of rRNA was followed by a robust G1 arrest, and activation of apoptotic proteins caspase-8, -9, and -3 and PARP-1 in a p53-independent manner. Using cell synchronization and flow cytometry, it was determined that cells treated while in G1 arrest immediately, but cells treated in S or G2 successfully complete mitosis. Twenty-four hours after treatment, the majority of cells finally arrest in G1, but nearly one-third contained highly compacted DNA; a distinct biological feature that cannot be associated with mitosis, senescence, or apoptosis. This unique effect mirrored the efficient condensation of tRNA and DNA in cell-free systems. The combination of DNA compaction and apoptosis by TriplatinNC treatment conferred striking activity in platinum-resistant and/or p53 mutant or null cell lines. Taken together, our results support that the biological activity of TriplatinNC reflects reduced metabolic deactivation (substitution-inert compound not reactive to sulfur nucleophiles), high cellular accumulation, and novel consequences of high-affinity noncovalent DNA binding, producing a new profile and a further shift in the structure–activity paradigms for antitumor complexes.

Substitution‐Inert Polynuclear Platinum Complexes as Metalloshielding Agents for Heparan Sulfate

“…Polyarginine cellular uptake is mediated by cell surface heparan sulfate proteoglycans (HSPGs) . The conceptualization of PPCs as “polyarginine mimics” led to the inhibition of the HSPG‐mediated cellular internalisation and nucleolar localization of the fluorescent‐labelled nona‐arginine peptide (TAMRA‐R 9 ) and identification of HSPGs as receptors for PPC cellular internalisation . In contrast to the mononuclear clinical platinum agents such as cisplatin and oxaliplatin, PPCs uniquely utilise this internalisation mechanism, which could provide an approach for selective uptake into tumours with high levels of HSPGs …”

Substitution‐Inert Polynuclear Platinum Complexes That Inhibit the Activity of DNA Polymerase in Triplex‐Forming Templates

“…[2] SI-PPCs display interesting biological activities including in vitro cytotoxicity in a wide range of cell lines, an antitumor activity that is independent of p53 status and high cellular accumulation. [1, 3] Rapid cellular entry is mediated through interaction with cell surface heparan sulfate proteoglycans (HSPG), which is a unique mechanism to SI-PPCs allowing for possibilities of tumor selectivity as well as disruption of HSPG function.…”

“…However, it has been suggested that it may be associated with high-affinity noncovalent binding to DNA resulting in very efficient nuclear condensation. [1, 3] This unique effect is mirrored by the efficient condensation of DNA and tRNA observed in vitro. [4] Previous studies also showed that SI-PPCs are potent inducers of B→A and B→Z conformational transitions in canonical sequences of DNA, they are even significantly more efficient than spermine.…”

Solution studies on DNA interactions of substitution-inert platinum complexes mediated via the phosphate clamp

“…The complex is cytotoxic at micromolar concentrations, similar to cisplatin, in a range of human tumor cell lines sensitive and resistant to cisplatin but is unaffected by serum degradation, unlike cisplatin or BBR3464. 33,34 Other notable features of the biological activity are the rapid nucleolar localization and nucleic acid condensation observed in cells . 34,35 The induction of apoptosis in tumor cells suggests that covalent Pt-DNA bond formation is not a prerequisite for antitumor activity for compounds with high DNA affinity, a further significant shift in the structure-activity paradigm of platinum antitumor agents.…”

“…33,34 Other notable features of the biological activity are the rapid nucleolar localization and nucleic acid condensation observed in cells . 34,35 The induction of apoptosis in tumor cells suggests that covalent Pt-DNA bond formation is not a prerequisite for antitumor activity for compounds with high DNA affinity, a further significant shift in the structure-activity paradigm of platinum antitumor agents. In conclusion our investigations emphasize the distinct modes of DNA binding by non-covalent polynuclear platinum complexes and the discrete nature of the phosphate clamp motif.…”