The Study On The Antitumor Activity And Mechanism Of Ruthenium(?) Complexes Containing Levofloxacin

Background and purpose:The World Health Organization's statistics on cancer in 2018 show that the incidence of malignant tumors has increased year by year.malignant tumors has become the most important factor that threatens human life in many countries and regions.With the development of natural sciences and basic disciplines,current anti-tumor treatment methods have been continuously enriched.Drug therapy has evolved from a single cytotoxic drug?chemotherapy?to endocrine,targeted,immunotherapy and other methods,which have significantly improved the survival of patients.At the same time,global cancer treatment-related costs continue to rise.According to statistics,expenditures for treatment and care rose from$96billion in 2013 to$133 billion in 2017,and cancer has become an important social and public health issue.Unfortunately,there are still many advanced patients who cannot benefit from other therapies,and chemotherapy is still their most important treatment.More effective,lower side effects,low cost,and broad-spectrum cytotoxic drugs are still the goals that scientists and clinicians are pursuing for their patients.Platinum-based compounds are currently the most widely used metal-based chemotherapeutics.It does not depend on the cell cycle and interferes with the DNA replication of cancer cells,leading to cancer cell apoptosis.However,its limited efficacy,poor tolerability,and large toxic and side effects limit its widespread application.Therefore,it is of great practical significance to develop new non-platinum metal drugs with higher efficiency and lower toxicity.At present,many research results have been made.Among them,ruthenium-based compounds show great anti-tumor potential,which is attributed to ruthenium compounds have good DNA binding properties,low toxicity,and the ability to produce different anti-tumor activities by changing their ligands.The most studied are divalent ruthenium-based compounds?II?and trivalent ruthenium-based compounds?III?.Studies have shown that metal complexes can improve their biological activity by using biologically active derivatives as ligands,Quinolones are widely used clinically as antibacterial drugs.they not only can combine with DNA/topoisomerase complex to kill bacteria in prokaryotes,but also can also combine with topoisomerase II of eukaryotic cells.Based on evidence of its targeting topoisomerase,many researchers used quinolones as ligands to synthesize metal complexes.Our research group found that ruthenium?II?–ofloxacin complexes with different bipyridine ligands can produces anticancer effects by binding to DNA.Levofloxacin and ofloxacin are both quinolone drugs,but there is still no research on the synthesis of new complexes with levofloxacin and polypyridine ruthenium complex.Two new ruthenium complexes[Ru?bpy?₂?LOFLX?]Cl·2H₂O?1?and[Ru?dmbpy?₂?LOFLX?]Cl·2H₂O?2?have been synthesized,and the antitumor activity and antitumor mechanism of the novel compound were explored through cell proliferation inhibition,DNA molecule binding,and DNA damage.Methods:1.levofloxacin,a biologically active organic compound,was used as the main ligand,and 2,2'-bipyridine?bpy? and 4,4'-dimethyl-2,2'-bipyridine?dmbpy?were used as auxiliary ligands to prepare two target ruthenium complexes[Ru?bpy?₂?LOFLX?]Cl·2H₂O?1?and[Ru?dmbpy?₂?LOFLX?]Cl·2H₂O?2?by using microwave-assisted synthesis technology.Their structures were characterized by ESI-MS,¹H NMR and ¹³C NMR.2.MTT analysis was used to evaluate the antitumor activity of complexes 1,2 and LOFLX against various tumor cells.3.Electron absorption spectroscopy,fluorescence spectroscopy,and viscosity experiments were used to study the molecular recognition of these two target ruthenium complexes with calf thymus DNA.The relationship between the affinity of ruthenium complexes for DNA and the antitumor activity of ruthenium complexes was studied.4.The apoptosis and cell cycle distribution were analyzed by flow cytometry.We use immunofluorescence experiments to investigate whether the target ruthenium complex can cause DNA damage.Results:?1?Two ruthenium complexes[Ru?bpy?₂?LOFLX?]Cl·2H₂O?1?and[Ru?dmbpy?₂?LOFLX?]Cl·2H₂O?2?were efficiently prepared by microwave-assisted synthesis.with yield of 63.6%and 65.8%,respectively.?2?The MTT assay shows that complex 2 exhibited acceptable inhibitory effect against human lung adenocarcinoma A549 cells,but with less toxicity toward HaCat normal human cells.?3?The DNA-binding behaviors show that LOFLX bind to DNA in electrostatic interaction modes,while complex 1 and 2 interacts with CT-DNA through a partial intercalative mode.Meanwhile,the DNA binding affinity follows the order2>1>LOFLX,which is consistent with their anticancer activities against A549 cells,indicating that DNA may be their potential target.?4?Flow cytometric assay showed that complex 2 effectively inhibits the proliferation of A549 cells by inducing G2/M phase arrest.Moreover,immunofluorescence analysis suggests that complex 2 can induce DNA damage in A549 cells as evidenced by the significant increase?H2AX foci formation.Conclusions:Taken together,these results suggest that target ruthenium complexes complex 2 using levofloxacin as the main ligand,and 2,2'-bipyridine?bpy?and 4,4'-dimethyl-2,2'-bipyridine?dmbpy?as auxiliary ligands exerts its inhibitory effect on the proliferation of A549 cells by inducing G2/M phase arrest through triggering DNA damage.As a potential anticancer drug,the complex 2 has great development and application prospects,and provides a basis for further research in the treatment of lung adenocarcinoma and other tumors.