Discovery of a Ruthenium Complex for the Theranosis of Glioma through Targeting the Mitochondrial DNA with Bioinformatic Methods

Abstract

Glioma is the most aggressive and lethal brain tumor in humans. Mutations of mitochondrial DNA (mtDNA) are commonly found in tumor cells and are closely associated with tumorigenesis and progress. However, glioma-specific inhibitors that reflect the unique feature of tumor cells are rare. Here we uncover RC-7, a ruthenium complex with strong red fluorescence, could bind with glioma mtDNA and then inhibited the growth of human glioma cells but not that of neuronal cells, liver, or endothelial cells. RC-7 significantly reduced energy production and increased the oxidative stress in the glioma cells. Administration of RC-7 into mice not only could be observed in the glioma mass of brain by fluorescence imaging, but also obviously prevented the growth of xenograft glioma and prolonged mouse survival days. The findings suggested the theranostic application of a novel type of complex through targeting the tumor mtDNA.

Keywords: computation docking; glioma; mtDNA mutation; organometallic complexes.

Figure 1

Chemical structures (A) and fluorescence characteristics of the RCs (RC-1~10). (B). The upper is the structures of RCs, and the below is the fluorescence of the RCs under visible or UV light.

Figure 2

Cell viability and concentration-effect relationship of complexes RCs with different types of cells at 50 nM and 12.5 nM. (A) Cell viability of U251MG human glioma cells, CATH.a neuronal cells, liver cells, and human endothelial cells after RC-7 addition. The values represent the mean ± SEM of the cell survival rate. The survival rate (%) was calculated as OD (sample–blank) / (control–blank) × 100%. (B) Dose-response curves of U251 and neuronal cell proliferation after RC-2, RC-3, RC-6, RC-7, and RC-9 were respectively added into the cell media at the concentration of 3.15~100 nM.

Figure 3

RC-7 induced U251MG cell apoptosis after arriving in mitochondria. (A) Intracellular distribution of RC-7 in U251MG cells. Cells were incubated with 50 nM RC-7 (red) for 2 h, followed by staining of mitochondria with MitoTracker green and the cells were observed with the confocal microscope. (B) Concentration-fluorescence intensity curve of RC-7 in U251MG cells. The fluorescence intensity increased in a RC-7 concentration-dependent manner. The data were expressed as mean ± SEM. Three independent experiments were performed. In addition, ATP (C), ROS (D), and GSH (E) were respectively determined in the RC-7 treated U251MG cells. (F) Cell apoptosis was measured by flow cytometry. The U251MG cells treated by RC-7 were collected and stained by Annexin V-FITC and DAPI for cell apoptosis assay.

Figure 4

Assessment of binding of RC-7 to glioma mtDNA. (A) Isolated mitochondria. The suspended and precipitated mitochondria under visible or UV light. (B) The mitochondria were observed under confocal microscope. The mitochondria showed red fluorescence under fluorescence excitation (Ex 550 nm). (C) RC-7 bound mtDNA rather than mitochondrial protein extract. The RC-7-mtDNA binding fluorescence profiles were evaluated by fluorescence (D) and UV spectroscopy (E). Concentrations of RC-7 from high to low were 10, 5, 2.5, 1.25, and 0.63 nM, respectively.

Figure 5

Binding of RCs and mtDNA fragments analyzed by agarose electrophoresis. (A) RCs were incubated with wild-type and mutant mtDNA fragments and then subjected to electrophoresis. RC-7 binding is highlighted in the rectangular box. (B) RC-7 (10, 5, 2.5, 1.25 nM) binding to wild-type and mutant D-loop mtDNA fragments. (C) The fluorescence intensity values of RC-7 binding with the dsDNA fragment containing different mutant sites were determined from three independent experiments, and the mean values were taken.

Figure 6

Molecular docking of RC-7 and mtDNA fragments. (A) Molecular docking of RC-7 with five mutant dsDNA (Table 1) by using PyMOL. (a) seq.1_mut. (b) seq.1_mut2. (c) seq.2_mut. (d) seq.3_mut. (e) seq.4_mut. (B) The binding affinities for RC-7 and four dsDNA sequences. Wild and mut respectively represent wild-type dsDNA and mutant dsDNA. ** p < 0.01 compared with the wild-type dsDNA. (C) RC-7 interacting with the mtDNA fragments such as (a) seq.1_wild. (b) seq.1_mut. (c) seq.1_mut2. The yellow dotted lines and values represent the three nearest distances (Å) between RC-7 and dsDNA mutation sites.

Figure 7

Bio-distribution of RC-7 in mice. (A) The distribution of RC-7 was detected with an in- vivo imaging system at 4 h after intravenous administration. (B) The distribution of RC-7 in important organs (the brain, liver, kidney, heart, spleen, and lungs). (C) Brain sections under a confocal microscope. The glioma mass showed strong red fluorescence at Ex 550 and Em 610.

Figure 8

Antitumor effect of RC-7 on glioma-bearing mice. (A) Glioma mass in striatum. Obvious glioma mass appeared about 3 weeks after U251MG cell microinjection. The arrows indicated the glioma mass. Representative images of HE staining and GFAP immunofluorescence in mouse brain sections (400 ×) from mice microinjected with saline and treated with saline (control), mice microinjected with U251MG and treated with saline (model), and mice microinjected with U251MG cells and treated with 10 mg/kg concentration of RC-7 daily at 12 days post-microinjection and 7 days post-treatment. (B) ATP content, (C) ROS, and (D) GSH levels in microinjection region. (E) Survival curve and (F) mean survival days of the tumor-bearing mice untreated or treated with RC-7 (n = 10 for each group). **p < 0.01 compared with the model mice.

Figure 9

The NMR spectra of RC-7. (A). ¹H-NMR spectrum. (B), ¹³C-NMR spectrum.