Studies On The Antitumor Activity And Mechanism Of Copper And Zinc Complexes

In this paper,we synthesized 11 Schiff based Zn and Cu complexes of thiosemicarbazides.The 2,6-diacetylpyridine or 2-acetyl-3-ethylpyrazine was condensed with thiosemicarbazides and its derivatives,and characterized by IR,elemental analysis,mass spectrometry,ESI,and single crystal X-ray methods.The interaction between Cu（II）complex and human serum albumin（HSA）was investigated by single crystal X-ray analysis.At the molecular level,the interaction between Cu（II）complex and G4 DNA was studied by UV and fluorescence.The cytotoxicity of Cu（II）,Zn（II）,and HSA-Cu（II）complexes,including apoptosis and autophagy were investigated by cellular experiments in vitro and used tumor nude mice for in vivo investigations.First,the five zinc（II）metal complexes were synthesized by the solution method with the formation of the thiosemicarbazone ligand and characterized by IR,ESI-MS,element analysis and single crystal X-ray analysis.Respectively as:[Zn（H₂DAPTsz）]₂（C1）,[Zn（H₂DAPTsz-Me）]₂（C2）,[Zn（H₂DAPTsz-tBu）]₂（C3）,[Zn（H₃DAPTsz-Ph）Cl]（C4）,[Zn（H₂DAPTsz-De）]₂（C5）.We obtained a bi-nuclear Zn（II）complex（C5）that showed significant cytotoxicity against human bladder cancer cells by multitargeting mechanism,including the blocking of the cell cycle in the S phase,inhibiting the cleavage of topoisomerase I（Topo I）,and inducing apoptosis and autophagy in T-24 cells.Besides,we determined their structure-activity relationships.Furthermore,C5 not only inhibited the migration of T-24 cell but also showed a significant cytostatic effect against the T-24 3D spheroid model.We synthesized six Cu（II）compounds derived from 2-Acetyl-3-ethylpyrazine-thiosemicarbazone（AepT）,and characterized by IR,ESI-MS,element analysis and single crystal X-ray analysis.Respectively as:[Cu（AepT）Cl]（C6）,[Cu（Aep4mT）Br]（C7）,Cu（Aep4pT）Br]（C8）,[Cu（Aep44mT）Br]（C9）,[Cu（Aep44eT）Br]（C10）,[Cu（AeppT）Br]（C11）.We investigated that these complexes penetrated the blood-brain barrier and inhibited the brain tumor growth and investigated their structure-activity relationships to brain tumor cells,obtaining a lead metal drug（C9）with remarkable cytotoxicity to glioma cell.Meanwhile,to improve the target ability and bioavailability of Cu compound in brain,we proposed to construct human serum albumin-Cu compound-penetrating peptide conjugate.The structure of the HSA-C9 complex revealed that C9 is bound to the IIA sub-domain of HSA.The His242 residual of HSA replaces the Br ligand in C9,coordinating with Cu²⁺.In vivo data showed that both C9 and HSA-C9-peptide conjugates penetrate the blood-brain barrier and inhibit brain tumor growth without any side effects.Furthermore,the HSA-C9-peptide conjugate also improves the bioavailability and therapeutic efficacy of C9 in vivo.In vitro data revealed that the HSA-Cu complexes improved the targeting ability and therapeutic efficiency of the Cu compounds.Moreover,Cu compounds and HSA-Cu complex exhibited cytotoxicity through multiple anticancer mechanisms,including arresting the cell cycle in S phase through the ROS-mediated intrinsic mitochondrial apoptosis,which accompanied by the regulation of Bcl-2 family protein.The C9 inhibited the topoisomerase I and topoisomerase II,stabilized the Pu27 G4 DNA,C9 and HSA-C9-peptide conjugates inhibited telomerase activity to promote apoptosis,and inhibited the migration and invasion of cells in a certain way.