Snythesis, Biological Evaluation And Action Mechanism Study Of Nonclassical Metal-based Anticancer Agents

Exploring novel chemotherapeutic agents is a great challenge in cancer medicine. Clinically used classical platinum-based anticancer agents, represented by cisplatin (cis-diamminedichloroplatinum(II)), are limited by side effects (such as myelosuppression, leucopenia, ototoxicity, neurotoxicity and nephrotoxicity) and drug resistance. To mitigate these drawbacks, two types of promising non-classical metal-based anticancer agents were studied by various methods.Platinum-acridines, represented by the prototypical Pt-Amidine, which are up to500-times more cytotoxic to non-small cell lung cancer cells than the clinical-used drug cisplatin, are very promising non-classcial metal-based anticancer agents. Adducts in nuclear DNA are the major cause of cancer cell death triggered by platinum-based anticancer drugs. Cellular uptake and accumulation, distribution and trafficking between subcellular compartments and, ultimately, localization to the nucleus are crucial parameters in the mechanism of platinum-based agents. Herein, high-performance liquid chromatography in conjunction with electrospray mass spectrometry (LC-ESMS) was used to structurally characterize the adducts formed by the Pt-Amidine in cell-free DNA. Pt-Amidine forms monofunctional adducts exclusively with guanine, based on the fragments identified in enzymatic digests (dG*, dGMP*, dApG*, and dTpG*, where the asterisk denotes bound drug). The time course of accumulation and DNA adduct formation of Pt-Amidine and the clinical drug cisplatin in NCI-H460lung cancer cells at physiologically relevant drug concentrations (0.1μM) was studied by inductively-coupled plasma mass spectrometry (ICP-MS). Pt-Amidine accumulates rapidly in cells and reaches intracellular levels of up to60-fold higher than those determined for cisplatin. The hybrid agent shows unusually high DNA binding levels:while cisplatin adducts form at a maximum frequency of5adducts per106nucleotides, Pt-Amidine produces25adducts per106nucleotides after only3h of continuous incubation with the lung cancer cells. The high overall levels of Pt-Amidine in the cells and in cellular DNA over the entire12-h treatment period translate into a rapid decrease in cell viability. In addition, to probe the subcellular distribution of Pt-acridine, an azide-modified derivative was synthesized and detected in treated NCI-H460cells using bioorthogonal fluorescent labeling. The results confirm high levels of platinum-acridine in the cells’nuclei and unexpected localization to the nucleoli.Based on oxidative DNA damage mechanism, six mononuclear Schiff Base copper complexes:[CuⅡ(5-Cl-pap)(OAc)(H2O)]·2H2O (1),[CuⅡ(5-Cl-pap)(H2O)2]NO3(2),[CuⅡ(pap)(H2O)2]NO3(3),[CuⅡ(5-NO2-pap)(H2O)2]ClO4-H2O (4),[Cu"(5-CH3-pap)(H2O)2]NO3(5),[Cu"(5-NO2-pap)(MeOH)(H2O)(NO3)](6),(5-X-pap=N-2-pyridi-yl-methylidene-2-hydroxy-5-X-phenylamine, X=H, Cl, CH3, NO2) were synthesized and characterized by X-ray diffraction. The interactions between complexes and calf thymus DNA were detected by spectroscopy methods. The apparent binding constants (Kapp=6.40x105,7.11x105,8.96×105,2.44×105,3.09×105,2.56×105M-1) of complexes indicated their moderate DNA affinity. Schiff Base copper complexes can induce strand breaks of pUC19plasmid DNA through an oxidative pathway, with singlet oxygen and hydrogen peroxide as reactive intermediates. Cytotoxicity studies in ten human cancer cell lines proved that Schiff Base copper complexes could inhibit the proliferation of cancer cells in a distinict manner from cisplatin.The results of nuclei staining by Hoechst33342and alkaline single-cell gel electrophoresis proved that complex3caused cellular DNA damage in HeLa cells. Furthermore, treatment of HeLa cells with3resulted in S-phase arrest, loss of mitochondrial potential, and up-regulation of caspase-3and-9in HeLa cells, suggesting that complex3was capable of inducing apoptosis in cancer cells through the intrinsic mitochondrial pathway. All these findings suggest mononuclear Schiff Base copper complexes as drug candidates for further development.