Comparison of the Effect of Native 1,4-Naphthoquinones Plumbagin, Menadione, and Lawsone on Viability, Redox Status, and Mitochondrial Functions of C6 Glioblastoma Cells

Abstract

Background: 1,4-naphthoquinones, especially juglone, are known for their anticancer activity. However, plumbagin, lawsone, and menadione have been less investigated for these properties. Therefore, we aimed to determine the effects of plumbagin, lawsone, and menadione on C6 glioblastoma cell viability, ROS production, and mitochondrial function.

Methods: Cell viability was assessed spectrophotometrically using metabolic activity method, and by fluorescent Hoechst/propidium iodide nuclear staining. ROS generation was measured fluorometrically using DCFH-DA. Oxygen uptake rates were recorded by the high-resolution respirometer Oxygraph-2k.

Results: Plumbagin and menadione displayed highly cytotoxic activity on C6 cells (IC₅₀ is 7.7 ± 0.28 μM and 9.6 ± 0.75 μM, respectively) and caused cell death by necrosis. Additionally, they increased the amount of intracellular ROS in a concentration-dependent manner. Moreover, even at very small concentrations (1-3 µM), these compounds significantly uncoupled mitochondrial oxidation from phosphorylation impairing energy production in cells. Lawsone had significantly lower viability decreasing and mitochondria-uncoupling effect, and exerted strong antioxidant activity.

Conclusions: Plumbagin and menadione exhibit strong prooxidant, mitochondrial oxidative phosphorylation uncoupling and cytotoxic activity. In contrast, lawsone demonstrates a moderate effect on C6 cell viability and mitochondrial functions, and possesses strong antioxidant properties.

Keywords: C6 glioma cell culture; lawsone; menadione; mitochondrial respiration; plumbagin; reactive oxygen species.

Figure 1

Structural formula of 1,4-naphthoquinones.

Figure 2

Effects of different concentrations of (A) plumbagin, (B) menadione, and (C) lawsone on the viability of C6 cells. Cell viability was assessed using (1) the MTT method and (2) double-staining with Hoechst 33342 and propidium iodide (PI). Hoechst 33342-positive cells, but lacking PI staining, were considered viable cells. PI-stained cells were considered necrotic. Data are presented as means of the percentage of the total cell number per micrograph ± SE (n = 5). * p < 0.05 versus control.

Figure 3

Effects of different concentrations of (A) plumbagin, (B) menadione, and (C) lawsone on intracellular ROS concentration. C6 cells were pre-treated with 10 μM DCFH-DA and then incubated with different concentrations of investigated compounds. Fluorescence intensity, which is proportional to the intracellular ROS concentration, was detected by a fluorometer at excitation and emission wavelengths of 544 and 590 nm, respectively. Data are presented as means of the percentage of control cells ± SE (n = 5). * p < 0.05 versus control.

Figure 4

Effects of different concentrations of plumbagin and ascorbate on the viability of C6 cells. C6 cells were treated with 5 µM and 15 µM concentrations of plumbagin and 100 µM and 250 µM of ascorbate for 24 h. Cell viability was assessed using the MTT method (for details, see the Materials and Methods). Data are presented as means of the percentage of the untreated control cells ± SE (n = 3). * p < 0.05 versus control.