Mithramycin forms a stable dimeric complex by chelating with Fe(II): DNA-interacting characteristics, cellular permeation and cytotoxicity

Abstract

Mith (mithramycin) forms a 2:1 stoichiometry drug-metal complex through the chelation with Fe(II) ion as studied using circular dichroism spectroscopy. The binding affinity between Mith and Fe(II) is much greater than other divalent metal ions, including Mg(II), Zn(II), Co(II), Ni(II) and Mn(II). The [(Mith)2-Fe(II)] complex binds to DNA and induces a conformational change of DNA. Kinetic analysis of surface plasmon resonance studies revealed that the [(Mith)2-Fe(II)] complex binds to DNA duplex with higher affinity compared with the [(Mith)2-Mg(II)] complex. A molecular model of the Mith-DNA-Metal(II) complex is presented. DNA-break assay showed that the [(Mith)2-Fe(II)] complex was capable of promoting the one-strand cleavage of plasmid DNA in the presence of hydrogen peroxide. Intracellular Fe(II) assays and fluorescence microscopy studies using K562 indicated that this dimer complex maintains its structural integrity and permeates into the inside of K562 cells, respectively. The [(Mith)2-Fe(II)] complex exhibited higher cytotoxicity than the drug alone in some cancer cell lines, probably related to its higher DNA-binding and cleavage activity. Evidences obtained in this study suggest that the biological effects caused by the [(Mith)2-Fe(II)] complex may be further explored in the future.

Figure 1

Chemical structure of mithramycin (Mith).

Figure 2

CD spectra of (A) Mith in the presence of Mg(II) with concentrations varying from 0 to 8 mM at 25°C (bottom). CD spectra of (B) Mith in the presence of Fe(II) with concentration varying from 0 to 0.1 mM (bottom) at 25°C. The drug concentration is 40 μM, buffered by 20 mM sodium-cacodylate at pH 7.3. The two isobestic points at 285 and 402 nm remain basically unchanged between the two complexes' association with Mg(II) and Fe(II) ions.

Figure 3

(A) CD titration of Mith against Fe(II) with normalized CD intensities change at 275 nm versus molar equivalents of [Fe(II)–Mith] at 25°C. The drug concentration is 40 μM, buffered by 20 mM sodium-cacodylate at pH 7.3. The two dashed lines in the figure represent the initial binding curve of Fe(II) to mithramycin and the one reaching the plateau. (B) Job-type titration plots for Fe(II)–Mith in 20 mM sodium-cacodylate at pH 7.3 at 25°C. The total concentration of Mith and metal was set at 60 μM.

Figure 4

(A) Comparison of the CD spectra of the [(Mith)₂–Fe(II)] complex, d(TTGGCCAA)₂, [(Mith)₂–Fe(II)]-d(TTGGCCAA)₂ complex and the sum of the CD spectra of [(Mith)₂–Fe(II)] plus d(TTGGCCAA)₂ in 20 mM sodium-cacodylate buffer at pH 7.3 with 100 mM NaCl at 25°C. (B) Sensorgram of Mith–DNA interaction between immobilized hairpin DNA duplex and the target [(Mith)₂–Fe(II)] and [(Mith)₂–Mg(II)] complexes by subtracting the reference of control. The concentration of each target is 10 μM, in 50 mM NaCl, buffered by 30 mM Tris–HCl (pH 7.3) at 20°C. The start (A) and end (B) point of the association and the start (C) and end (D) point of the dissociation are indicated by arrows.

Figure 5

The effects of EDTA and Mith on integrity of the supercoiled plasmid DNA (pET-21b) when incubated in the presence of Fe(II) and hydrogen peroxide. The ratios of drugs versus Fe(II) are represented as 5×, 1× and 0.1× for 5:1, 1:1 and 1:10, respectively. The assays were carried out as described in Materials and Methods.

Figure 6

(A) The effect of the [(Mith)₂–Fe(II)] complex on calcein-detectable cellular Fe(II) content. The fluorescence of calcein-loaded K562 cells were treated with two different compounds as denoted by arrows, including 30 μM FeCl₂ (control), and 10 μM [(Mith)₂–Fe(II)] complex, buffered by PBS buffer at pH 7.3. (B) Subcellular locations of the [(Mith)₂–Fe(II)] complex are detected by phase contrast (left) and fluorescence (right) microscope. The location of cell nucleus was indicated by arrows.

Figure 7

(A) The drawings of the metal(II)-coordinated [Mith-(TTGGCCAA)]₂ model viewed from the minor groove (left) and backbone direction, Mith containing chromophore (yellow) and saccharide (green) in ball and stick, and DNA in skeletal line drawing. (B) Surface representation of the metal(II)-coordinated [Mith-(TTGGCCAA)]₂ model shown with DNA electrostatic potentials viewed from the minor groove (left) and backbone (right). The metal(II) and waters are depicted in pink and blue, respectively.