The novel ruthenium—γ‐linolenic complex [Ru₂(aGLA)₄Cl] inhibits C6 rat glioma cell proliferation and induces changes in mitochondrial membrane potential, increased reactive oxygen species generation and apoptosis in vitro

Abstract

The present study reports the synthesis of a novel compound with the formula [Ru(2)(aGLA)4Cl] according to elemental analyses data, referred to as Ru(2)GLA. The electronic spectra of Ru(2)GLA is typical of a mixed valent diruthenium(II,III) carboxylate. Ru(2)GLA was synthesized with the aim of combining and possibly improving the anti-tumour properties of the two active components ruthenium and gamma-linolenic acid (GLA). The properties of Ru(2)GLA were tested in C6 rat glioma cells by analysing cell number, viability, lipid droplet formation, apoptosis, cell cycle distribution, mitochondrial membrane potential and reactive oxygen species. Ru(2)GLA inhibited cell proliferation in a time and concentration dependent manner. Nile Red staining suggested that Ru(2)GLA enters the cells and ICP-AES elemental analysis found an increase in ruthenium from <0.02 to 425 mg/Kg in treated cells. The sub-G1 apoptotic cell population was increased by Ru(2)GLA (22 +/- 5.2%) when analysed by FACS and this was confirmed by Hoechst staining of nuclei. Mitochondrial membrane potential was decreased in the presence of Ru(2)GLA (44 +/- 2.3%). In contrast, the cells which maintained a high mitochondrial membrane potential had an increase (18 +/- 1.5%) in reactive oxygen species generation. Both decreased mitochondrial membrane potential and increased reactive oxygen species generation may be involved in triggering apoptosis in Ru(2)GLA exposed cells. The EC(50) for Ru(2)GLA decreased with increasing time of exposure from 285 microM at 24 h, 211 microM at 48 h to 81 microM at 72 h. In conclusion, Ru(2)GLA is a novel drug with antiproliferative properties in C6 glioma cells and is a potential candidate for novel therapies in gliomas.

Inhibition of C6 rat glioma proliferation by [Ru2Cl(Ibp)4] depends on changes in p21, p27, Bax/Bcl2 ratio and mitochondrial membrane potential

“…This is highly suggestive that the RuIbp complex causes intracellular stress which leads to cell cycle arrest and subsequent low level increases in apoptosis. Similar effects have been reported for another ruthenium complex containing gamma-linolenic acid in glioma cells [14,27]. In human colon carcinoma cells ibuprofen was found to cause an increase in p53 protein expression concomitant with an increase in Bax protein expression [28].…”

“…These properties were analyzed by FACS and demonstrated important metabolic changes induced by RuIbp after 72 h treatment. The mitochondrial membrane potential in C6 cells typically exhibits two populations one with high and one with low potential [14,16]. RuIbp decreased the number of high potential mitochondria by only approximately 5%, but the average mitochondrial membrane potential was significantly decreased by approximately 27% as seen in the green FACS channel (FLH-1).…”

“…The effects of 100 μM RuIbp were compared to control cells receiving only ethanol vehicle at 1% final concentration. These concentrations were chosen based on previous studies in tumor cells [10,14].…”

“…The cells were collected by trypsinization, washed in PBS and pooled with free-floating cells collected from the culture medium and fixed in 4% formaldehyde, freshly prepared from paraformaldehyde, in 0.1 M potassium phosphate, pH 7.2. The fixed cells were pelleted, resuspended in 0.1 mg/mL RNase A in PBS and incubated at 37°C for 2 h. After pelleting, the cells were resuspended in 20 μg/mL propidium iodide in PBS and maintained on ice before measuring the fluorescence on a Beckton Dickinson FACS [14].…”

“…The cells were collected by trypsinization, washed (×4) with PBS, pH 7.2 and maintained in PBS on ice before measuring the fluorescence on a Beckton Dickinson FACS [14].…”