Investigation Of Anticancer Mechanisms For Platinum Complexes Based On Pt-DNA Biological Probes

As one of the most widely used anticancer drugs in clinic, cisplatin binds to DNA and forms mainly 1,2-d(G*pG*) and 1,2-d(A*pG*) intra-strand cross-links and less frequently 1,3-d(G*pTpG*) cross-links and inter-strand cross-links. These cisplatin-DNA adducts can be recognized by several cellular proteins (HMGB1) and consequently initiate a series of cellular events such as blocking DNA replication and gene transcription, triggering diverse signal pathways, and eventually lead to apoptosis or systematic cell death. To plot the entire network contributing to cytotoxicity to cisplatin, platinated DNA have been employed as probes to capture proteins which interacting with Pt-DNA adducts. Though several proteins have been identified and comfirmed to recognizing Pt-DNA adducts, there are still several key questions remain to be answered. Novel protein candidates other than HMGB proteins are required to mapping the entire pharmacological complexity of platinum drugs. Moreover, the precise role of HMG recognition of Pt-DNA adducts is still obscure although it has been extensively studied.Therefore, a general tool, which can capture proteins that bind to cisplatin-DNA adducts, is currently lacking. Such a probe will be useful for understanding the recongnition mechanisms of protein and damaged DNA. It may also be useful to provide profound insight of cellular self-defense systems and cisplatin-resistance.Here we report a kind of peptide-oligonucleotide conjugates (POC) based biomolecular probes (P1-4, TP1-5) which are designed for investigating the interactions between Pt-DNA crosslink and their binding partners under biological condition. In these probes, ploy-His peptides which act as immobilization hook and oligonucleotides which contain site specific Pt-crosslink and act as protein binding bait are covalently linked with heterobifunctional cross-linker. The major intention of this probe is to mimic native interactions of Pt-DNA-protein ternary complexes and to isolate them intact from cell extract. With none platinum modified POC as control probe, we established a well-defined control panel which could discriminate proteins contributing to Pt-DNA adducts recognition rather than general DNA-binding proteins. Based on well-developed proteomics techniques, a number of proteins candidates can be identified.For probe P1, there is no surprise that HMGB1/2 is identified as the most prominent cisplatin-DNA binding protein. Besides, several other proteins factors like SFPQ/NONO, hnRNP DO are also newly found. We are surprised to find that most of HMGB1/2 proteins are found as posttranslational modification (PTM) forms with hyper-acetylation and specific phosphorylation. There are at least 4 kinds of PTM isoforms of HMGB1 identified in this study. The isoform A and B are found globally acetylated, and isoform C and D possess 3 phosphorylation sites respectively in addition to acetylation. The latter two phosphorylation patterns of HMGB1 have not been detected previously. Possible biological relevance of this discovery may root in local conformation change after phosphorylation. Increased binding and bending ability of phosphorylated HMGB1 could facilitate exposure of damaged DNA toward repair proteins or other signal factors. Nevertheless, cellular events after the recognition are still not clear. Studies on downstream molecular mechanisms are required in the future.Comparison of proteins captured by probes P2-4 and P1 reveals that the ability of HMGB1 recognizing Pt-DNA lesions is obviously influenced by different carrier ligands of these platinum complexes. We investigate probes of P2-4, which correspond to platinum compounds include oxaliplatin, PtCl2[1(S),2(S)-DACH], and PtCl2(o-phenylenediamine), in pulling down proteins from cell extract. Oxaliplatin and PtCl2[1(S),2(S)-DACH] possess a carrier ligand which exhibits more steric hindrance than cisplatin. The hydrophobicity is also increased dramatically compared to cisplatin. As to PtCl2(o-phenylenediamine), introducing of benzene ring not only changes the coordination environment for platinum, but also elevates the steric hindrance and hydrophobicity. Our results indicate that affinity of HMGB 1 and DNA distorted by platinum complexes are decreasing when the steric hindrance and hydrophobicity of their carrier ligands is increasing.Moreover, same protocol has been successfully applied to polynuclear Pt complexes we synthesized before. Probes TP1-4 are constructed based on DNA-Pt adducts of trinuclear platinum complexes-[Pt3(HPTAB)Cl3](C104)3 and MTPC. These two different compounds are preferentially to form long range crosslinks with DNA and display potent cytotoxicity and distinct cellular mechanism from cisplatin. Proteins captured by these probes include transcription factor YBOX1, SFPQ, Annexin A2, DNA binding protein SSBP and Heterogeneous nuclear ribonucleoproteins A2/B1. The well-known DNA damage recognition protein HMGB proteins are not found upon these probes. This may explain the very different pharmacology of cisplatin and trinuclear platinum complexes. These results will inspire further extensive studies and lead to better understanding on overall molecular pathways of platinum drugs.