| Isothermal titration calorimetry (ITC), viscosity measurement and some spectroscopic methods (UV-vis titration, steady-state fluorescence emission quenching experiment and circular dichroism spectroscopy) as well as some molecular biological methods (equilibrium dialysis, gel electrophoresis) have been used to comparatively investigate the interactions of calf thymus DNA (CT DNA) and yeast tRNA with a new synthesized Ru(II) complex [Ru(dmp)2PMIP]2+, which is designed basing on our previous research. MTT assay and trypan blue assay are applied to test the cytotoxity of the Ru(II) complex to human lung tumor cells of A549. Then flow cytometry experiments are used to test whether the cytotoxic Ru(II) complex induces apoptosis of human lung cancer cell line A549. Furthermore, we perform the experiments to find whether Ru(II) complex promotes the ability of cleaving plasmid pBR322 DNA, and we elucidate the possible cell apoptosis mechanism of the Ru(II) complex. The concrete points of the report are as below:(1) A new Ru(II) complex [Ru(dmp)2PMIP]2+ (dmp = 2,9-dimethyl-1,10-phenanthroline, PMIP= 2-(4-methylphenyl)imidazo[4,5-f] 1,10-phenanthroline) has been designed and synthesized, which has the same intercalated ligand and different ancillary ligand with the previous investigated Ru(II) complex [Ru(phen)2PMIP]2+(phen= 1,10-phenanthroline) by our group. Viscosity measurement and some spectroscopic methods (UV-vis titration, steady-state fluorescence emission quenching experiment and circular dichroism spectroscopy) as well as some molecular biological methods (equilibrium dialysis, gel electrophoresis) have been used to comparatively investigate the interactions of the Ru(II) complex [Ru(dmp)2PMIP]2+ with calf thymus DNA (CT DNA) and yeast tRNA. The results of spectroscopic methods and viscosity measurement show that the Ru(II) complex [Ru(dmp)2PMIP]2+ has a moderately strong binding affinity to both CT DNA and yeast tRNA with a binding mode of intercalation, while CT DNA binding of the complex is stronger than that of yeast tRNA. Equilibrium dialysis and CD results indicate that both interactions are enantioselective and the△enantiomer of the complex may bind more favorably to both CT DNA and yeast tRNA than the A enantiomer does, while enantioselective binding ability of CT DNA with the complex is stronger than that of yeast tRNA. The results suggest that the structure of nucleic acids could be related to their interactions with Ru(II) complex.(2) The Ru(II) complex [Ru(phen)2PMIP]2+ (phen-1,10-phenanthroline, PMIP= 2-(4-methylphenyl)imidazo[4,5-f] 1,10-phenanthroline) has been synthesized. Isothermal titration calorimetry (ITC) technique has been used to comparatively investigate the interactions of the Ru(II) complex [Ru(dmp)2PMIP]2+ and [Ru(phen)2PMIP]2+ with calf thymus DNA (CT DNA)/yeast tRNA. The CT DNA binding of the complex [Ru(dmp)2PMIP]2+ is stronger than that of yeast tRNA at any temperatures examined. The binding of the complex [Ru(dmp)2PMIP]2+ to CT DNA is driven by a moderately favorable enthalpy decrease with a moderately favorable entropy increase, while the interaction of the complex with yeast tRNA is driven by a large favorable enthalpy decrease in combination with a less favorable entropy increase at lower temperatures and driven by a large favorable enthalpy decrease only at higher temperatures. Both complex [Ru(dmp)2PMIP]2+ and [Ru(phen)2PMIP]2+ intercalate into the paired double strand areas of CT DNA and yeast tRNA by a single set of identical sites model. The binding affinities of complex [Ru(dmp)2PMIP]2+ to CT DNA/yeast tRNA are weaker than those of complex [Ru(phen)2PMIP]2+ due to the influence of ancillary ligand. Negative molar heat capacity changes of the binding reactions demonstrate the burial of hydrophobic and hydrophilic solvent-accessible surface of CT DNA/yeast tRNA on complexation with both Ru(II) complexes.(3) Cell viability experiments including MTT assay and trypan blue assay indicate the Ru(II) complex shows significant dose-dependent cytotoxicity to human lung tumor cells of A549. Further flow cytometry experiments show that the cytotoxic Ru(II) complex induces apoptosis of human lung cancer cell line A549. Upon irradiation at 365 nm, the Ru(II) complex is found to form the singlet oxygen and promote the cleavage of plasmid pBR322 DNA from supercoiled form I to nicked form II, exhibiting time-dependent single-strand cleavage. Our data demonstrate that the Ru(II) polypyridyl complex binds to DNA, and showed DNA cleavage ability, resulting in DNA damage and the subsequent cell apoptosis in tumor cells, suggesting that anti-tumor activity of the Ru(II) complex could be related to its interaction with DNA.The studies shown above will be useful for the application in designing chemical nucleases and nucleic acid-targeting drugs based on understanding of the structures of metal complexes in the future. |
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