Fig. 1.

The melatonin-shikonin (Mel-SHK) combination exerts strong anti-cancer activities and downregulates SIRT3/SOD2 in U937 cells. U937 cells were treated with melatonin (Mel, 0–2 mM) or shikonin (SHK, 0–0.9 μM) alone or in combination for 6 h unless otherwise indicated. (A) DNA fragmentation was assessed. The histogram depicts the percentage of DNA fragmentation in cells treated with SHK alone (white bars) or in combination with Mel at various concentrations. The effects of SHK (0.75 μM) and concomitant dose-dependent effect of Mel (0–2 mM) were investigated with respect to (B) DNA fragmentation, (C) cell viability, (D) Combination Index (CI), (E) apoptosis [Annexin V/propidium iodide (PI) double-staining] and (F) hypodiploid (sub-G1) DNA content. (G) Cells with high levels of reactive oxygen species (ROS) were evaluated at 1 h and 3 h post-treatment. (H) SOD2 and SIRT3 protein expression levels were analysed by western blotting 1 h after treatment with Mel and SHK alone or in combination. The bar graph depicts the relative expression levels compared to SHK treatment alone. (I) Relative analysis of SOD2 activity. (J) Mitochondrial calcium (Ca²⁺) levels at 1 h and (K) mitochondrial membrane potential (MMP) at 4 h, 5 h and 6 h post-treatment. (L) Western blotting analysis of pro-apoptotic and anti-apoptotic protein expression. Flow cytometry data are presented as histograms and bar graphs. Relative protein expression was calculated for all western blots, using actin for normalisation and as a loading control. One representative figure from at least three independent experiments is shown for each assay. The data in each bar graph are presented as the means ± standard errors of the means (SEM). *P < 0.05, **P < 0.01, and ***P < 0.001 relative to SHK treatment alone.