Study On Mechanism Of Tumor Cell Apoptosis Induced By Quercetin Metal Complexes And Interaction Of Complexes With DNA

In recent years malignant tumor has been one of the main causations of death, and antitumor drug screening has been a significant task in pharmaceutics. It was reported that both antioxidant activity and tumor cell proliferation inhibitory effect of some quercetin metal complexes were stronger than those of quercetin alone. However, little attention is paid to antitumor mechanisms of quercetin metal complexe. So in order to elucidating antitumor mechanism of quercetin metal complexes, we focus our study on the effect of quercetin metal complexes(Mn, Co, Ni, Cu, Zn) on tumor cell apoptosis, and their DNA-binding and cleavage activity. Based on these, we discussed the mechanism of cell apoptosis induced by quercetin metal complexes and the interaction mechanism of the complexes with DNA.1) Antitumor activity of quercetin metal complexes and HepG2 cell apoptosis induced by them was studied using MTT assay, fluorescence staining, flow cytometry (FCM) and immunocytochemical method. And the molecular mechanisms of tumor cell apoptosis induced by quercetin metal complexes are discussed preliminarily.i) It is found that quercetin metal complexes have a significant inhibition to growth and proliferation of HepG2, SMMC-7721 and A549 cell lines in a concentration- and time-dependent manner. Furthermore, the proliferation inhibitory effect of quercetin manganese, nickel and zinc complexes on tumor cells is stronger than that of quercetin alone. After tumor cells were treated by quercetin manganese, nickel and zinc complexes for 48h,the half inhibitory concentration (IC₅₀) were 20.64,14.44 and 10.67μmol·L^-1 respectively, while IC₅₀ of quercetin was 33.69μmol·L^-1ii) After treatment with quercetin manganese, nickel and zinc complexes, morphological changes of HepG2 cells apoptosis were observed with fluorescence microscope. Results indicate that they could induce HepG2 cells apoptosis.iii) FCM results suggest that HepG2 cells are arrested at G₀/G₁ after treatment with quercetin manganese, nickel and zinc complexes, which could contribute to tumor cell apoptosis.iv) Evidence is provided that expression levels of bc1-2 and survivin protein are down-regulated and expression level of p53 protein is up-regulated after treatment with quercetin manganese, nickel and zinc complexes, which activates caspase-3 resulting in HepG2 cells apoptosis. Down-regulation of survivin and bcl-2 could exert both synergistic effect and complementation effect in HepG2 cell apoptosis induced by quercetin metal complexes. Therefore HepG2 cells apoptosis induced by quercetin manganese, nickel and zinc complexes is not regulated by single factor, but rather by multiple pathways.2) The interaction of quercetin metal complexes with calf thymus(CT) DNA is investigated by electronic absorption spectroscopy, fluorescence spectroscopy, viscosity measurement. Plasmid pBR322 DNA cleavage is also studied by gel electrophoresis. Furthermore, DNA-binding properties and mechanism of DNA cleavage are discussed preliminarily. We put forward the nuclease-liked activity of quercetin metal complexes for the first time.i) Results infer that quercetin manganese, cobalt, nickel and zinc complexes could interact with DNA through intercalation mode, but DNA-binding of quercetin copper complexes is more than one mode, including intercalation and electrostatic mode.ii) Results suggest that quercetin metal complexes could cleave supercoiled pBR322 DNA to nicked and line form effectively. Among them, DNA cleavage by quercetin copper complex is the most efficient. Moreover, low ionic strength could promote DNA strand breakage induced by quercetin manganese, nickel, copper and zinc complexes, while high ionic strength could prevent the formation of double strand breaks. However, increasing ionic strength could only promote DNA cleavage induced by quercetin cobalt complex. So DNA cleavage induced by quercetin metal complexes depends on ionic strength in the solution.iii) The decrease of supercoiled DNA induced by quercetin metal complexes with time shows the expected exponential nature of the curves, which confirms the process to pseudo-first-order. Their rate constants are 1.18×10^-4s^-1(Mn), 0.53×10^-4s^-1(Co), 0.76×10^-4s^-1(Ni),4.82×10^-4s^-1(Cu),1.68×10^-4s^-1(Zn), respectively.iv) Results imply that the process of DNA cleavage by quercetin manganese, cobalt, nickel and zinc complexes occurs via a hydrolytic path, while the process of DNA cleavage by quercetin copper complex occurs via a redox path involved in Cu(â… )OOH.3) Mechanisms of antitumor activity and interaction of quercetin metal complexes with DNA were elucidated theoretically by quantum chemistry and molecular mechanics methods.i) Based on quantum chemistry calculation of the structure of quercetin metal complexes, it is found that freakish octahedron structure comprises six oxygen atoms and a metal ion, which has good stability. Furthermore, coordinate properties of quercetin metal complexes are discussed through nature population analysis and frontier orbital analysis, which confirms the coordinate action of hydroxyl oxygen is stronger than that of carbonyl oxygen.ii) The calculation results suggest that the energies of LUMO and NLUMO of quercetin metal complexes are lower than the energies of the HOMO and NHOMO for the CG/GC stacking in DNA backbones, which contributes to enhance theÏ€-Ï€interaction between complexes and DNA base-pairs. The antitumor activity and DNA-binding properties of quercetin metal complexes could rely heavily on their LUMO energies and total charge of ligand of complexes.iii) The processes of quercetin metal complexes intercalating into DNA are studied by molecular modeling. Results infer that quercetin metal complexes are inclined to intercalate into base stack in CG/GC region from minor groove, which depends on asymmetric charge distribution and steric interaction. Base on calculated potential energy of quercetin metal complexes intercalating into DNA base stack, it is suggested that calculated DNA-binding action decreases in the order Zn(Que)₂>Ni(Que)₂>Mn(Que)₂ >Co(Que)₂>Cu(Que)₂,according with experiment results. It is proposed that quercetin metal complexes may inhibit transcription of survivin promoter with abundant GC,which contributes to down-regulating expression of survivin protein and promote tumor cell apoptosis.In the present paper, molecular mechanisms of tumor cells apoptosis induced by quercetin metal complexes and mechanisms of interaction of the complexes with DNA are discussed, which lay the foundation of development of new efficient chemical nuclease and antitumor drug, and provide a new consideration for novel antitumor drug design.