ABSTRACT
Chromomycin (Chro) forms a 2:1 drug/metal complex through the chelation with Fe(II), Co(II), or Cu(II) ion. The effects of spermine on the interaction of Fe(II), Co(II), and Cu(II) complexes of dimeric Chro with DNA were studied. Circular dichroism (CD) measurements revealed that spermine strongly competed for the Fe(II) and Cu(II) cations in dimeric Chro-DNA complexes, and disrupted the structures of these complexes. However, the DNA-Co(II)(Chro)(2) complex showed extreme resistance to spermine-mediated competition for the Co(II) cation. According to surface plasmon resonance (SPR) experiments, a 6mM concentration of spermine completely abolished the DNA-binding activity of Fe(II)(Chro)(2) and Cu(II)(Chro)(2) and interfered with the associative binding of Co(II)(Chro)(2) complexes to DNA duplexes, but only slightly affected dissociation. In DNA integrity assays, lower concentrations of spermine (1 and 2mM) promoted DNA strand cleavage by Cu(II)(Chro)(2), whereas various concentrations of spermine protected plasmid DNA from damage caused by either Co(II)(Chro)(2) or Fe(II)(Chro)(2). Additionally, DNA condensation was observed in the reactions of DNA, spermine, and Fe(II)(Chro)(2). Despite the fact that Cu(II)(Chro)(2) and Fe(II)(Chro)(2) demonstrated lower DNA-binding activity than Co(II)(Chro)(2) in the absence of spermine, while Cu(II)(Chro)(2) and Fe(II)(Chro)(2) exhibited greater cytoxicity against HepG2 cells than Co(II)(Chro)(2), possibly due to competition of spermine for Fe(II) or Cu(II) in the dimeric Chro complex in the nucleus of the cancer cells. Our results should have significant relevance to future developments in metalloantibiotics for cancer therapy.
Subject(s)
Antibiotics, Antineoplastic/metabolism , Chromomycin A3/metabolism , Coordination Complexes/metabolism , DNA/metabolism , Spermine/metabolism , Antibiotics, Antineoplastic/chemistry , Cell Line, Tumor , Chromomycin A3/chemistry , Cobalt/metabolism , Coordination Complexes/chemistry , Copper/metabolism , Dimerization , Humans , Iron/metabolismABSTRACT
Few studies have examined the effects of polyamines on the action of DNA-binding anticancer drugs. Here, a Co(II)-mediated dimeric mithramycin (Mith) complex, (Mith)(2)-Co(II), was shown to be resistant to polyamine competition toward the divalent metal ion when compared to the Fe(II)-mediated drug complexes. Surface plasmon resonance experiments demonstrated that polyamines interfered with the binding capacity and association rates of (Mith)(2)-Co(II) binding to DNA duplexes, while the dissociation rates were not affected. Although (Mith)(2)-Co(II) exhibited the highest oxidative activity under physiological conditions (pH 7.3 and 37 degrees C), polyamines (spermine in particular) inhibited the DNA cleavage activity of the (Mith)(2)-Co(II) in a concentration-dependent manner. Depletion of intracellular polyamines by methylglyoxal bis(guanylhydrazone) (MGBG) enhanced the sensitivity of A549 lung cancer cells to (Mith)(2)-Co(II), most likely due to the decreased intracellular effect of polyamines on the action of (Mith)(2)-Co(II). Our study suggests a novel method for enhancing the anticancer activity of DNA-binding metalloantibiotics through polyamine depletion.
Subject(s)
Antibiotics, Antineoplastic/toxicity , Cell Survival/drug effects , Cobalt/toxicity , DNA, Bacterial/metabolism , Dimerization , Plicamycin/toxicity , Spermidine/pharmacology , Spermine/pharmacology , Antibiotics, Antineoplastic/metabolism , Antibiotics, Antineoplastic/therapeutic use , Binding, Competitive , Cell Line, Tumor , Circular Dichroism , Cobalt/metabolism , Cobalt/therapeutic use , DNA, Bacterial/antagonists & inhibitors , DNA, Superhelical/antagonists & inhibitors , DNA, Superhelical/metabolism , Humans , Mitoguazone/toxicity , Plicamycin/antagonists & inhibitors , Plicamycin/therapeutic use , Spermidine/antagonists & inhibitors , Spermine/antagonists & inhibitorsABSTRACT
Chromomycin A3 (Chro) has been evidenced to exhibit much higher binding affinity toward Fe(II) by forming a highly stable 2:1 drug/metal complex, compared to its structural analogue, mithramycin (Mith). Different properties of the [(Chro)2-Fe(II)] complex acting on DNA, such as sequence specificity, DNA cleavage, and topoisomerase I (TopI) inhibition were studied. Kinetic analyses of surface plasmon resonance showed that the affinity of the [(Chro)2-Fe(II)] complex upon binding to hairpin DNA duplexes containing various tetranucleotide sequences follows the order: GGCC > CGCG > CCGG approximately GCGC > AGCT > ACGT > TGCA > TCGA. According to circular dichroism (CD) studies, most hairpin DNA duplexes appeared to retain their B-type conformations in the presence of the [(Chro)2-Fe(II)] complex, except the duplex containing the GGCC sequence, which exhibited the features of both A- and B-type DNA. In DNA-cleavage assays, the [(Chro) 2-Fe(II)] complex was shown to cause single-stranded cleavage of plasmid DNA because of a Fenton-type reaction. DNA cleavage activity of the [(Chro) 2-Fe(II)] complex was increased at low pH. Moreover, the complex was capable of inhibiting TopI activity. The [(Chro)2-Fe(II)] complex exhibited higher cytotoxicity than the [(Mith) 2-Fe(II)] complex in several cancer cell lines, most likely owing to its more stable dimeric structure and higher DNA-binding affinity. Our results provide significant evidence that the [(Chro)2-Fe(II)] complex could be promising in terms of its biological applications in the future.
