Shikonin Directly Targets Mitochondria and Causes Mitochondrial Dysfunction in Cancer Cells

Figure 2

Induction of ROS, oxidative DNA damage, and elevated intracellular Ca²⁺ levels by shikonin. (a) Flow cytometric analysis of ROS levels after treatment with different concentrations of shikonin for 1 h or 50 μM H₂O₂ for 15 min in living U937 cells. Cells were stained with H₂DCFH-DA and measured at 488 nm excitation and detected using a 530/30 nm bandpass filter. (b) Statistical quantification of ROS induction after shikonin treatment in U937 cells. Data points represent mean (fold change) ± SEM of at least three independent experiments. (c) ROS induction kinetics in live cells. U937 cells were stained with H₂DCFH-DA and ROS induction was measured by flow cytometry. After 2 min, shikonin was added to the cells and measurement was continued for 1 h. Shikonin was excited at 640 nm and detected with a 730/45 nm bandpass filter. DCF was excited with a 488 nm laser and detected using a 530/30 nm bandpass filter. (d) Induction of DNA damage by shikonin measured using alkaline elution technique. Columns indicate the number of DNA single-strand breaks (SSB) and of Fpg-sensitive modifications (oxidative DNA damage) after shikonin treatment. Data points represent mean ± SEM of at least three independent experiments. (e) Real-time kinetics of intracellular Ca²⁺ levels after treatment with different concentrations of shikonin or ionomycin in U937 cells. Cells were stained with indo-1 and [Ca²⁺]_𝑖 was measured by flow cytometry. After 2 min, shikonin was added to the cells and measurement was continued for 1 h. Indo-1 was excited with a 355 nm laser and the ratio of the signals detected using a 405/20 nm filter and a 530/30 filter (405/20 nm/530/30 nm) was used as an index for intracellular calcium concentration (*significant difference according to Student’s 𝑡-test, 𝑃<0.05).