Preparation,DNA Binding And Anticancer Activities Of Four Novel Oxyvanadium Complexes In Vitro And In Vivo

Background and objective:Malignant tumor,a disease with the highest mortality rate at present,seriously threaten human health.Metallic antitumor drugs,represented by platinum,have the advantages of wide spectrum and high efficiency,which are still one of the research hotspots of chemotherapy drugs.In recent years,it has been found that the vanadium complexes have the advantages of low toxicity and strong anti-tumor activity,especially the anti-tumor potential of oxovanadium complexes has been widely concerned.In this paper,four novel oxyvanadium complexes are synthesized,and their interaction with DNA is discussed.The effects of complexes on the proliferation in cancer cell lines in vitro and nude mice xenografts in vivo were evaluated,and their anti-tumor mechanism is discussed.The present study will provide a new experimental evidence for the development of oxovanadium complexes as a potential anti-tumor drug.Methods:Four oxovanadium phenanthroimidazole complexes with different substituents were synthesized and Elemental analysis,MS,IR and NMR spectroscopy were implemented to characterize the complexes and ligands.The interaction modes of these complexes with calf-thymus DNA(CT-DNA)were investigated using UV-vis absorption,fluorescence spectra,viscosity measurements and gel electrophoresis.The anti-proliferation effects of these complexes and ligands on seven human cancer cell lines(human gastric cancer SGC-7901 cells,human cervical adenocarcinoma HeLa cells,human bladder cancer BIU-87 cells,human lung cancer SPC-A-1 cells,human liver cancer HepG2 cells,human colon cancer HT-29 cells and human pancreatic cancer PANC-1 cells)and IC50 values were analyzed by MTT assay.The cell cycle distribution was analyzed by flow cytometry with PI staining.Apoptosis was measured by flow cytometry with Annexin V-FITC/PI double staining.Detection of intracellular ROS level was carried out by fluorescent probe DCFH-DA.Morphological changes to cell nuclei were identified after Hoechst 33258 staining.Mitochondrial membrane depolarization(MMP)was analyzed by JC-1 dye staining.Expression of apoptosis related-proteins(Bax?Bcl-2?Cyt-C?Cleaved caspase-3?Cleaved caspase-8 and Cleaved caspase-9)and cell cycle regulatory proteins(p16?Cyclin D1?CDK4 and p-Rb)were measured by western blot.The median lethal dose(LD50)of the complex was calculated by Bliss method through acute toxicity experiment in mice,and histopathological changes were observed by Hematoxylin-Eosin(H-E)staining.Nude mice xenografts were established by inoculation of HeLa cells,and the weight and the tumor volume of xenograft nude mice were measured on0 d,7 d,14 d,21 d and 28 d after tumor transplantation,respectively.In vivo imaging system were performed on nude mice xenografts on 7 d and 28 d.The apoptosis of HeLa cells in tumor tissue was detected by TUNEL(FITC)+DAPI double staining,and the expression of Ki-67 and p16 in tumor tissue was detected by immunohistochemistry.Results:Four oxovanadium phenanthroimidazole complexes VO(hntdtsc)NPIP,VO(hntdtsc)CPIP,VO(hntdtsc)MEPIP and VO(hntdtsc)HPIP have been synthesized and characterized.All four oxovanadium complexes can bind with CT-DNA with an intercalation model and can also efficiently cleave plasmid pBR322 DNA.The K_b values of DNA-binding affinities are as follows:VO(hntdtsc)NPIP>VO(hntdtsc)CPIP>VO(hntdtsc)MEPIP>VO(hntdtsc)HPIP.Four oxovanadium complexes showed certain inhibition on the proliferation of seven human cancer cell lines,and VO(hntdtsc)NPIP showed the largest inhibitory effects against the Hela,BIU-87,SPC-A-1,SGC-7901,HT-29,PANC-1,HepG2 and SPC-A-1 cell lines with IC50 values of 1.09±0.16,4.51±0.68,7.61±0.55,14.23±1.36,26.34±4.11,35.23±6.25,19.32±3.45?M,respectively.The proliferation inhibition of VO(hntdtsc)NPIP on Hela cells is stronger than that of cisplatin,and the half maximal inhibitory concentration(IC50)value is one-fifth of that of cisplatin(5.54±0.81?M).The proliferation inhibition rate of 24,48 and 72 hours incubation of VO(hntdtsc)NPIP(0.5,1.0,2.0?M)on Hela cell were:24 h:(10.37±1.01)%,(22.55±3.23)%and(34.52±4.61)%;48 h:(35.33±3.36)%,(45.30±7.23)%and(57.46±6.69)%;72 h:(42.36±7.33)%,(60.44±8.63)%and(74.61±7.81)%.The number of cells at G0/G1 phase in HeLa cells was significantly increased(F=22.03,P<0.01),and the proportion of S-phase cells was decreased(F=16.14,P<0.05).VO(hntdtsc)NPIP significantly increased the apoptosis rate with a manner of dose-dependent in HeLa cells(F=43.65,P<0.001).Hoechst 33258 staining demonstrated that Hela cells showed significant nuclear fragmentation,dense nuclear concentration,and strong blue fluorescence after 48 h incubation of complex.VO(hntdtsc)NPIP significantly increased the ROS level in HeLa cells(F=112.24,P<0.001).JC-1 staining showed that VO(hntdtsc)NPIP increased the green fluorescence strength in HeLa cells(F=55.22,P<0.01),which indicates a decrease in the mitochondrial membrane potential of HeLa cells.The down-regulation of the protein levels for Bcl-2(F=33.12,P<0.001),Cyclin D1(F=19.15,P<0.05),CDK4(F=20.41,P<0.05),p-Rb(F=75.46,P<0.001)and Bcl-2/Bax ratio(F=86.74,P<0.001)with the simultaneous up-regulation of the Bax(F=16.22,P<0.05),Cyt-C(F=54.32,P<0.001),Cleaved Caspase-3(F=25.54,P<0.01),Cleaved Caspase-8(F=34.68,P<0.001),Cleaved Caspase-9(F=40.63,P<0.001)and p16(F=68.55,P<0.001)protein expressions were observed in HeLa cells after VO(hntdtsc)NPIP treatment.Acute toxicity tests in mice showed that the LD50 of VO(hntdtsc)NPIP was24.26 mg/kg,and there was no significant toxicity to the heart,liver,kidneys and lung tissues of mice with a dose of 4.0 mg/kg.VO(hntdtsc)NPIP(1.0,2.0,4.0 mg/kg)can dose-dependently inhibited the increase of tumor volume of HeLa xenografts in nude mice(F=134.60,P<0.001),and reduced relative mean optical density of HeLa xenografts in nude mice in vivo imaging system on 28 d after tumor transplantation(F=89.65,P<0.001).TUNEL(FITC)+DAPI double staining results showed that VO(hntdtsc)NPIP increased the green fluorescence/blue fluorescence ratio dose-dependently(F=133.14,P<0.001),which increased the apoptosis rate of HeLa cells in tumor tissue.VO(hntdtsc)NPIP significantly reduced the Ki-67 staining positive cell rate(F=140.22,P<0.001),and increased p16 staining positive cell rate(F=79.55,P<0.01).Conclusion:All four oxovanadium complexes showed DNA binding and anti-proliferation activities in human cancer cells,and VO(hntdtsc)NPIP showed the strongest activity.VO(hntdtsc)NPIP can inhibit proliferation of human cervical cancer HeLa cells in vitro and in vivo.Our results suggest that the anti-proliferation activity of VO(hntdtsc)NPIP might be associated with the induction of G0/G1 phase cell cycle arrest through p16-Cyclin D1-CDK4-p-Rb pathway and apoptosis via caspase-dependent mitochondrial pathway.